Circ-0000953 deficiency exacerbates podocyte injury and autophagy disorder by targeting Mir665-3p-Atg4b in diabetic nephropathy.

Circular RNAs (circRNAs) are special non-coding RNA (ncRNA) molecules that play a significant role in many diseases. However, the biogenesis and regulation of circRNAs in diabetic nephropathy (DN) are largely unknown. Here, we investigated the expression profile of circRNAs in kidney of DN mice through circular RNA sequencing (circRNA-seq). The renal biopsy samples of patients with DN had low circ -0,000,953 expression, which was significantly associated with renal function. Furthermore, loss-of-function and gain-of-function experiments were carried out to prove the role of circ -0,000,953 in DN. Podocyte conditional knockin (cKI) or systemic overexpression of circ -0,000,953 alleviated albuminuria and restored macroautophagy/autophagy in kidney of diabetic mice. However, circ -0,000,953 knockdown exacerbated albuminuria and podocyte injury. Mechanistically, we found circ -0,000,953 directly binds to Mir665-3p-Atg4b to perform its function. Silencing of Mir665-3p or overexpression of Atg4b recovered podocyte autophagy both in vitro and in vivo. To examine the cause of circ -0,000,953 downregulation in DN, bioinformatics prediction found that circ -0,000,953 sequence has a high possibility of containing an m6A methylation site. Additionally, METTL3 was proved to regulate the expression and methylation level of circ -0,000,953 through YTHDF2 (YTH N6-methyladenosine RNA binding protein 2). In conclusion, this study revealed that circ -0,000,953 regulates podocyte autophagy by targeting Mir665-3p-Atg4b in DN. Therefore, circ -0,000,953 is a potential biomarker for prevention and cure of DN.Abbreviation: CCL2/MCP-1: C-C motif chemokine ligand 2; ceRNA: competing endogenous RNA; circRNA: circular RNA; cKI: conditional knockin; cKO: conditional knockout; CRE: creatinine; DM: diabetes mellitus; DN: diabetic nephropathy; ESRD: end-stage renal disease; HG: high glucose; IF: immunofluorescence; MAP1LC3/LC3B: microtubule-associated protein 1 light chain 3 beta; MPC5: mouse podocyte clone 5; MTECs: mouse tubular epithelial cells; MTOR: mechanistic target of rapamycin kinase; NC: normal control; ncRNA: non-coding RNA; NPHS1: nephrosis 1, nephrin; NPHS2: nephrosis 2, podocin; PAS: periodic acid-Schiff; RELA/p65: v-rel reticuloendotheliosis viral oncogene homolog A (avian); SDs: slit diaphragm proteins; Seq: sequencing; STZ: streptozotocin; SV40: SV40-MES13-cells, mouse mesangial cell line; T1D: type 1 diabetes mellitus; T2D: type 2 diabetes mellitus; TEM: transmission electron microscopy; TNF/TNF-α: tumor necrosis factor; VECs: vascular endothelial cells; WT1: WT1 transcription factor; YTHDF2: YTH N6-methyladenosine RNA binding protein 2.

Paeoniflorin alleviates ischemia/reperfusion induced acute kidney injury by inhibiting Slc7a11-mediated ferroptosis.

The pathophysiological mechanism of acute kidney injury (AKI) is complicated, and effective drugs are still lacking. Ferroptosis is a newly discovered regulatory cell death mode characterized by the lethal accumulation of iron and reactive oxygen species-(ROS-)-dependent lipid hydroperoxides. In recent years, ferroptosis has been confirmed to be involved in the progression of AKI. Paeoniflorin (PF) is a traditional Chinese medicine that has protective effects on a variety of kidney diseases including AKI. However, the mechanism by which PF attenuates AKI is unclear. We detected that PF attenuated serum biochemical markers, histological damage, ferroptosis and inflammation in a dose-dependent manner in a mouse AKI model with bilateral renal artery ischemia-reperfusion (IR). Hypoxia-reoxygenation (HR)-induced ferroptosis and inflammation was also inhibited by PF in human renal tubular epithelial cells (HK2). RNA sequence analysis revealed that PF inhibited ferroptosis in HK2 cells by upregulating Slc7a11 in the glutathione pathway after HR treatment. PF failed to further protect cells with specific knockdown of Slc7a11 from ferroptosis under HR conditions. Consequently, these data indicated that PF prevention of ferroptosis in AKI requires dependence on Slc7a11. This study provided a scientific basis for the clinical search for drugs to prevent IR induced AKI.

Paeoniflorin binds to VEGFR2 to restore autophagy and inhibit apoptosis for podocyte protection in diabetic kidney disease through PI3K-AKT signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Paeoniflorin (PF) was found to exhibit renal protection from diabetic kidney disease (DKD) in previous trials, but its specific mechanism remains to be elucidated. AIM OF THE STUDY: This study furtherly explored the specific mechanism of PF in protect podocyte injury in DKD. MATERIALS AND METHODS: We observed the effects of PF on renal tissue and podocytes in DKD by constructing the vitro and vivo models after measuring the pharmacokinetic characteristics of PF. Target proteins of PF were found through target prediction, and verified by molecular docking, CESTA, and SPR, and then furtherly explored the downstream regulation mechanism related to podocyte autophagy and apoptosis by network prediction and co-immunoprecipitation. Finally, by using the target protein inhibitor in vivo and knocking down the target protein gene in vitro, it was verified that PF played a role in regulating autophagy and apoptosis through the target protein in diabetic nephropathy. RESULTS: This study found that in STZ-induced mice model, PF could improve the renal biochemical and pathological damage and podocyte injure (p < 0.05), upregulate autophagy activity (p < 0.05), but inhibit apoptosis (p < 0.01). Vascular endothelial growth factor receptor 2 (VEGFR2), predicted as the target of PF, directly bind with PF reflected by molecular docking and surface plasmon resonance detection. Animal studies demonstrated that VEGFR2 inhibitors have a protective effect similar to that of PF on DKD. Network prediction and co-immunoprecipitation further confirmed that VEGFR2 was able to bind PIK3CA to regulate PI3K-AKT signaling pathway. Furthermore, PF downregulated the phosphorylation of PI3K and AKT (p < 0.05). In vitro, similarly to autophagy inhibitors, PF was also found to improve podocyte markers (p < 0.05) and autophagy activity (p < 0.05), decrease caspase 3 protein (p < 0.05) and further inhibited VEGFR2-PI3K-AKT activity (p < 0.05). Finally, the results of VEGFR2 knockdown were similar to the effect of PF in HG-stimulated podocytes. CONCLUSION: In conclusion, PF restores autophagy and inhibits apoptosis by targeting the VEGFR2-mediated PI3K-AKT pathway to improve renal injury in DKD, that provided a theoretical basis for PF treatment in DKD.

Paeoniflorin directly binds to TNFR1 to regulate podocyte necroptosis in diabetic kidney disease.

Necroptosis was elevated in both tubulointerstitial and glomerular renal tissue in patients with diabetic kidney disease (DKD), and was most pronounced on glomerulus in the stage with macroalbuminuria. This study further explored whether paeoniflorin (PF) could affect podocyte necroptosis to protect kidney injure in vivo and in vitro. Our study firstly verified that there are obvious necroptosis-related changes in the glomeruli of DKD through bioinformatics analysis combined with clinicopathological data. STZ-induced mouse diabetes model and high-glucose induced podocyte injury model were used to evaluate the renoprotection, podocyte injury protection and necroptosis regulation of PF in DKD. Subsequently, the target protein-TNFR1 that PF acted on podocytes was found by computer target prediction, and then molecular docking and Surface plasmon resonance (SPR) experiments were performed to verify that PF had the ability to directly bind to TNFR1 protein. Finally, knockdown of TNFR1 on podocytes in vitro verified that PF mainly regulated the programmed necrosis of podocytes induced by high glucose through TNFR1. In conclusion, PF can directly bind and promote the degradation of TNFR1 in podocytes and then regulate the RIPK1/RIPK3 signaling pathway to affect necroptosis, thus preventing podocyte injury in DKD. Thus, TNFR1 may be used as a new potential target to treat DKD.

Melatonin Alleviates Acute Kidney Injury by Inhibiting NRF2/Slc7a11 Axis-Mediated Ferroptosis.

Acute kidney injury (AKI) is still a puzzling clinical problem; its pathophysiology is not completely understood. Up to now, an effective treatment for AKI is lacking. Ferroptosis is a novel form of regulated cell death characterized by the lethal accumulation of lipid hydroperoxides that are dependent on iron and reactive oxygen species and mitochondrial dysfunction. Recently, ferroptosis was shown to play a vital role in AKI such as ischemia-reperfusion kidney injury and folic acid-induced AKI. Melatonin (MT) is an antioxidant that regulates the sleep-wake cycle. While the therapeutic effect of melatonin on AKI has been reported, its mechanism for the treatment of renal ferroptosis remains unclear. We found that melatonin treatment significantly alleviated the serum biochemistry index and histopathological alterations in vivo AKI models induced by bilateral renal artery ischemia reperfusion and folic acid in mice. Ferroptosis induced by hypoxia and reoxygenation or erastin (Era) in mouse tubular epithelial cells (MTEC) was also rescued by melatonin treatment. RNA sequence analysis of ferroptosis-related genes showed that melatonin affects oxidative stress responses by inhibiting hypoxia and reoxygenation- (HR-) mediated downregulation of NRF2 and upregulation of Slc7a11 in MTEC. Specific knockdown of NRF2 increased the sensitivity of cells to ferroptosis, and melatonin failed to protect against ferroptosis in the HR condition. Together, our data indicate that melatonin prevents ferroptosis in AKI by acting on the NRF2/Slc7a11 axis.

Clinical Significance of Glomerular Autophagy in Evaluation of Diabetic Kidney Disease Progression.

Background: Diabetic kidney disease (DKD) is closely associated with the death or survival of resident kidney cells. Aim: The purpose of this study was to determine the changes in renal cell survival and death in DKD and their diagnostic values in DKD progression. Materials and Methods: This study analyzed a dataset of renal tissues from DKD patients to identify changes in genes associated with renal cell death and survival. Our findings were subsequently validated in human kidney tissues. Differential indicators of DKD patients' clinicopathological data screened by stepwise regression and glomerular P62 protein expression were included in binary logistic regression analysis to assess the impact of these parameters on DKD progression. A receiver operating characteristic (ROC) curve analysis was employed to evaluate the diagnostic value of P62 protein in DKD progression. Results: Bioinformatics analysis results revealed that glomerular autophagy in DKD was more significantly altered, which was consistent with the semi-quantitative results of P62 in glomeruli. Further studies established that P62 expression was mainly increased in podocytes. Stepwise regression analysis indicated that changes in the expressions of glomerular P62 and apolipoprotein A1 (ApoA1) might be involved in the progression of DKD. However, binary logistic regression analysis results suggested that only P62 was significantly associated with DKD development. ROC curve analysis showed that the area under the curve (AUC) of P62 for the detection of DKD was 0.905. Conclusion: Autophagy inhibition occurred in both glomeruli and tubules, and was most pronounced in glomerular podocytes. The levels of P62 protein in glomeruli, as an autophagy activity indicator, was one of the predictors of entering the stage of macroalbuminuria in DKD.

Identification of Genes Reveals the Mechanism of Cell Ferroptosis in Diabetic Nephropathy.

Aims/Introduction: Diabetic nephropathy (DN) is one of the main complications of diabetes. Genomics may reveal the essential pathogenesis of DN. We analyzed datasets to search for key genes to explore pathological mechanisms of DN. Materials and Methods: In this study, weighted gene co-expression network analysis (WGCNA) was used to divide the differential expression genes (DEGs) from GSE142025 into different modules, and enrichment pathway analysis was conducted for each module to find key genes related to cell death pathway. Then, verification was carried out through network and histopathology. Finally, the regulatory mechanisms of key gene expression, including transcription factors (TFs), miRNA and E3 ligases related to ubiquitination, were predicted through website prediction and then miRNA results were validated using GSE51674 dataset. Results: The results of WGCNA and enrichment pathway analysis indicated that ferroptosis had significantly occurred in advanced DN (AND) group. Analysis of DEGs indicated that the occurrence and development of ferroptosis are mainly through ALOX15-mediated lipid metabolism pathway, which was found in all intrinsic cells of the glomerulus detected by IHC and IF staining. Moreover, network predictions were used for searching ALOX15-related TFs and ubiquitination. Meanwhile, the network predictions combining with other dataset furtherly discovered miRNAs which regulated ALOX15 expression. This study showed that the levels of mmu-miR-142-3p increased in DN mice kidney tissues, compared with the NC group. Conclusion: Ferroptosis existed in glomerular intrinsic cells of ADN group and its potential key candidate gene was ALOX15 which may be regulated by miR-142 and miRNA-650, TFs (CREBBP, EP300, HDAC1, MTA1, SPI1, STAT6) and E3 ligases related to ubiquitination (PML, ZMIZ1, MARCHF1, MARCHF3, MARCHF8, MARCHF11).

A novel small molecule Hsp90 inhibitor, C-316-1, attenuates acute kidney injury by suppressing RIPK1-mediated inflammation and necroptosis.

Acute kidney injury (AKI) is marked by a fast deterioration of the kidney function that may be caused by a variety of factors. Recently, although our group found that PPBICA alleviated programmed cell death in AKI, poor water solubility limited its bioavailability. In this research, we screened a series of derivatives and found that C-316-1 had the best suppressive effect on preventing necroptosis and inflammation in cisplatin- and ischemia/reperfusion-induced AKI in vitro and in vivo with lower toxicity and better water solubility. Mass spectrometry results showed that C-316-1 bound to heat shock protein 90 (Hsp90), which was further confirmed by molecular docking and surface plasmon resonance. Additionally, the Hsp90 expression was upregulated in the blood and tissues of AKI patients. We discovered that C-316-1 decreased the RIPK1 protein level without affecting its mRNA expression. The proteasome inhibitor, MG132 restored the level of RIPK1 reduced by C-316-1, suggesting that C-316-1 limits necroptosis by promoting the degradation of RIPK1 rather than by reducing its production. Immunoprecipitation further showed that pretreatment with C-316-1 disrupted the Hsp90-Cdc37 protein-protein Interactions (PPIs). Thereby, C-316-1 inhibited the Hsp90-Cdc37 complex formation and led to a significant decrease in RIPK1, which in turn reduced necroptosis. Moreover, C-316-1 treatment did not protect against kidney injury in vivo and in vitro when Hsp90 was knocked down and R46, E47, and S50 in Cdc37 binding site of Hsp90 might form an important active pocket with C-316-1. These findings suggest that C-316-1 is a potential therapeutic agent against RIPK1-Mediated Necroptosis in AKI.

Risk factors for Encapsulating Peritoneal Sclerosis in patients undergoing peritoneal dialysis: A meta-analysis.

PURPOSE: Encapsulating Peritoneal Sclerosis (EPS) is the most serious complication of long-term peritoneal dialysis (PD), which considerably reduces the patient's quality of life, leading to patients discontinuing PD. Considering these negative effects, it is necessary to systematically review and determine the risk factors of EPS. METHODS: The PubMed, Embase, Web of Science, Cochrane Library, and China Biology Medicine (CBM) were searched from their inception to January 1st, 2022, and the bibliographies from the citations of relevant articles were manually searched. The ROBINS-I (Risk of Bias in Non-randomized studies of Interventions) tool was used to evaluate the risk of bias of included studies. RESULTS: Ten studies involving 12595 participants were included in this meta-analysis. The results revealed that a younger age at PD onset (MD = -7.70, 95% CI, -11.53~-3.86), a higher transporter (MD = 0.13, 95% CI, 0.09~0.18), a longer PD duration (SMD = 1.15, 95% CI, 0.68~1.61), a longer peritonitis duration (MD = 12.66, 95% CI, 3.85~21.47), and history of glomerulonephritis (OR = 1.42, 95% CI, 1.02~1.97) were significant risk factors for EPS. However, sex, use of icodextrin, the number of peritonitis episodes, and history of multicystic kidney disease did not affect the risk of EPS. CONCLUSIONS: This review provides a scientific basis for further understanding the etiology of PD-related EPS and improving prevention strategies. More high-quality studies are necessary to validate this paper's findings.

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 in vitro. 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 in vivo 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.

Carnosine alleviates podocyte injury in diabetic nephropathy by targeting caspase-1-mediated pyroptosis.

Diabetic nephropathy (DN) is a main complication of diabetes and often develops into end-stage nephropathy. Histologically, DN progresses as the gradual loss of podocytes with the loss of glomerular podocytes being the earliest sign of DN. Pyroptosis is a new type of programmed cell death and has been mechanistically correlated with podocyte injury in DN. The current study aimed to evaluate the protective effects of carnosine on glomerular podocytes in DN, both in vivo and in vitro. Using high glucose-treated cultured MPC5 cells and a streptozotocin (STZ)-induced diabetic mouse model, we evaluated the effects of carnosine on alleviating podocyte injury in DN. We found that carnosine significantly reversed albuminuria and histopathological lesions and alleviated renal inflammatory and pyroptosis responses in STZ-induced diabetic mice for 12 weeks. The results also showed that carnosine strongly inhibited podocyte inflammation and podocyte pyroptosis in vitro. Cellular Thermal Shift Assay (CETSA) and molecular docking results revealed that mechnaistically caspase-1 was the target of carnosine. We then found that silencing caspase-1 eliminated the protective effect of carnosine. Interestingly, we also found that caspase-1 and gasdermin D expression were increased in renal biopsy tissue of patients with DN. Our study is the first to demonstrate the novel role of carnosine in alleviating podocyte injury by inhibiting pyroptosis via the targeting of caspase-1. Carnosine may have potential as a therapeutic agent in treating DN by targeting caspase-1.

Carnosine alleviates diabetic nephropathy by targeting GNMT, a key enzyme mediating renal inflammation and fibrosis.

Diabetic nephropathy (DN) is a common microvascular complication of diabetes and the main cause of end-stage nephropathy (ESRD). Inflammation and fibrosis play key roles in the development and progression of diabetic nephropathy. By using in vivo and in vitro DN models, our laboratory has identified the protective role of carnosine (CAR) on renal tubules. Our results showed that carnosine restored the onset and clinical symptoms as well as renal tubular injury in DN. Furthermore, carnosine decreased kidney inflammation and fibrosis in DN mice. These results were consistent with high glucose (HG)-treated mice tubular epithelial cells (MTECs). Using web-prediction algorithms, cellular thermal shift assay (CETSA) and molecular docking, we identified glycine N-methyltransferase (GNMT) as a carnosine target. Importantly, we found that GNMT, a multiple functional protein that regulates the cellular pool of methyl groups by controlling the ratio of S-adenosylmethionine (SAM) to S-adenosylhomocysteine (SAH), was down-regulated significantly in the serum of Type 1 DM patients and renal tissues of DN mice. Moreover, using cultured TECs, we confirmed that the increased GNMT expression by transient transfection mimicked the protective role of carnosine in reducing inflammation and fibrosis. Conversely, the inhibition of GNMT expression abolished the protective effects of carnosine. In conclusion, carnosine might serve as a promising therapeutic agent for DN and GNMT might be a potential therapeutic target for DN.

TAB1 regulates glycolysis and activation of macrophages in diabetic nephropathy.

OBJECTIVE AND DESIGN: Macrophages exhibit strong phenotypic plasticity and can mediate renal inflammation by polarizing into an M1 phenotype. They play a pivotal role in diabetic nephropathy (DN). Here, we have investigated the regulatory role of transforming growth factor ß-activated kinase 1-binding protein 1 (TAB1) in glycolysis and activation of macrophages during DN. METHODS: TAB1 was inhibited using siRNA in high glucose (HG)-stimulated bone marrow-derived macrophages (BMMs) and lentiviral vector-mediated TAB1 knockdown was used in streptozotocin (STZ)-induced diabetic mice. Western blotting, flow cytometry, qRT-PCR, ELISA, PAS staining and immunohistochemical staining were used for assessment of TAB1/nuclear factor-κB (NF-κB)/hypoxia-inducible factor-1α (HIF-1α), iNOS, glycolysis, inflammation and the clinical and pathological manifestations of diabetic nephropathy. RESULTS: We found that TAB1/NF-κB/HIF-1α, iNOS and glycolysis were up-regulated in BMMs under HG conditions, leading to release of further inflammatory factors, Downregulation of TAB1 could inhibit glycolysis/polarization of macrophages and inflammation in vivo and in vitro. Furthermore, albuminuria, the tubulointerstitial damage index and glomerular mesangial expansion index of STZ-induced diabetic nephropathy mice were decreased by TAB1 knockdown. CONCLUSIONS: Our results suggest that the TAB1/NF-κB/HIF-1α signaling pathway regulates glycolysis and activation of macrophages in DN.