Dapagliflozin attenuates ferroptosis in diabetic nephropathy through activation of the Nrf2/HO‑1 signaling pathway.

NOD2 promotes renal injury by exacerbating inflammation and podocyte insulin resistance in diabetic nephropathy

The present study aimed to evaluate the role and mechanism of ferrostatin-1 (Fer-1) in oxalate (Ox)-induced renal tubular epithelial cell injury, fibrosis, and calcium oxalate (CaOx) stone formation. A CaOx model in mice kidneys was established via intraperitoneal injection of 80 mg/kg glyoxylic acid for 14 days. The mice were randomly divided into three groups (n=6), namely, the control (Con), the CaOx group, and the CaOx + Fer-1 group. Cultured human renal tubular epithelial cells (HK-2 cells) were randomly divided into three groups (n=3), namely, the control (Con), the Ox group, and the Ox + Fer-1 group. The levels of heme oxygenase 1 (HO-1), superoxide dismutase 2 (SOD2), glutathione peroxidase 4 (GPX4), and solute carrier family 7 member 11 (SLC7A11) were assessed by immunofluorescence and western blot analysis. Renal tubular injury and apoptosis were evaluated by H&E and TUNEL staining. Kidney interstitial fibrosis was evaluated by Masson and Sirius red staining, and the levels of E-cadherin, vimentin and α-SMA were detected by immunofluorescence or western blot analysis. Mitochondrial structure was observed using a transmission electron microscope. The levels of reactive oxygen species (ROS) were determined by flow cytometry and CaOx stone formation was evaluated by von Kossa staining. The results revealed that in comparison with the Con group, mitochondrial injury under glyoxylic acid treatment was observed by TEM. The expression of GPX4 and SLC7A11 in the CaOx and Ox groups was downregulated (P<0.05), whereas the expression of HO-1 and SOD2 was upregulated (P<0.05). Renal tissue damage, apoptosis of renal tubular epithelial cells, and interstitial fibrosis were increased in the CaOx and Ox groups (P<0.05). In comparison with the CaOx or Ox group, the expression of GPX4 and SLC7A11 in the CaOx + Fer-1 or Ox + Fer-1 group was upregulated (P<0.05), whereas that of HO-1 and SOD2 was downregulated (P<0.05). Renal tissue damage, apoptosis of renal tubular epithelial cells and interstitial fibrosis were decreased following Fer-1 treatment (P<0.05). The ROS level was also decreased following Fer-1 treatment. Moreover, CaOx stone formation was decreased in the CaOx + Fer-1 group (P<0.05). In conclusion, Fer-1 alleviated Ox-induced renal tubular epithelial cell injury, fibrosis, and CaOx stone formation by inhibiting ferroptosis.

Hyperglycemia-induced renal tubular cell injury is thought to play a critical role in the pathogenesis of diabetic nephropathy (DN). However, the role of miRNAs in renal tubular cell injury remains to be fully elucidated. The aim of the present study was to investigate the role and mechanisms of miRNAs protecting against high glucose (HG)-induced apoptosis and inflammation in renal tubular cells. First, we analyzed microRNA (miRNA) expression profiles in kidney tissues from DN patients using miRNA microarray. It was observed that miRNA-140-5p (miR-140-5p) was significantly down-regulated in kidney tissues from patients with DN. An inverse correlation between miR-140-5p expression levels with serum proteinuria was observed in DN patients, suggesting miR-140-5p may be involved in the progression of DN. HG-induced injury in HK-2 cells was used to explore the potential role of miR-140-5p in DN. We found that miR-140-5p overexpression improved HG-induced cell injury, as evidenced by the enhancement of cell viability, and inhibition of the activity of caspase-3 and reactive oxygen species (ROS) generation. It was also observed that up-regulation of miR-140-5p suppressed HG induced the expressions of pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), interleukin (IL)-1β and IL-6 in HK-2 cells. In addition, TLR4, one of the upstream molecules of NF-κB signaling pathway, was found to be a direct target of miR-140-5p in the HK-2. Moreover, the HG-induced activation of NF-κB signaling pathway was inhibited by miR-140-5p overexpression. These results indicated that miR-140-5p protected HK-2 cells against HG-induced injury through blocking the TLR4/NF-κB pathway, and miR-140-5p may be considered as a potential prognostic biomarker and therapeutic target in the treatment of DN.

Patulin (PAT) is a common mycotoxin, widely found in cereals, seafood, nuts, and especially in fruits and their products. Exposure to this mycotoxin has been reported to induce kidney injury. However, the possible mechanism remains unclear. In our study, short-term high-dose intake of PAT caused acute kidney injury (AKI) in mice. We performed high-throughput transcriptional sequencing to identify differentially expressed genes (DEGs) between the treatment and control groups. The ferroptosis signaling pathway had the highest enrichment, suggesting ferroptosis is involved in PAT-induced AKI. Further, the existence of ferroptosis and autophagy was confirmed by observing the changes of mitochondria morphology and the formation of autophagosomes by electron microscopy. And the expression of solute carrier family 7 member 11 (SLC7A11), glutathione peroxidase 4 (GPX4), p62, nuclear receptor coactivator 4 (NCOA4), and ferritin heavy chain 1 (FTH1) were downregulated, whereas acyl-CoA synthase long-chain family member 4 (ACSL4), transferrin (TF), LC3, and ferritin light chain (FTL) expression were upregulated in PAT-exposed mice. These results suggested autophagy-dependent ferroptosis occurred in the animal model. This view has also been confirmed in the human renal tubular epithelial cell (HKC) experiments. Autophagy inhibitor 3-methyladenine (3MA) attenuated PAT-induced ferroptosis and the iron contents in HKC cells. Simultaneous autophagy-dependent ferroptosis can be inhibited by ferroptosis inhibitors ferrostatin-1 (Fer-1) and desferrioxamine (DFO). In general, this study provides a new perspective for exploring the new mechanism of acute kidney injury caused by PAT.

Aims: Diabetic nephropathy (DN) is a major cause of end-stage renal disease, with no therapeutic interventions available to control its progression. Ferroptosis, an iron-dependent regulated cell death characterized by lipid peroxidation, plays a pivotal role in the pathogenesis of DN. Human umbilical cord mesenchymal stem cells (hUCMSCs) are an effective treatment modality for DN; however, the underlying mechanism of action remains unclear. The aim of the present study was to investigate whether hUCMSCs alleviate DN via inhibiting ferroptosis and its molecular mechanisms in type 2 diabetic mice and high-glucose and palmitate-stimulated human renal tubular epithelial cell (HK-11) models. Results: Our findings revealed that hUCMSCs improved the renal structure and function and tubular injuries. HUCMSC treatment can inhibit ferroptosis by decreasing iron content, reducing reactive oxygen species, malondialdehyde and 4-hydroxynonenal generation, decreasing the expression of positive ferroptosis mediator transferrin receptor 1 and long-chain acyl-CoA synthetase 4, and enhancing the expression of negative ferroptosis mediators (i.e., ferritin heavy chain, glutathione peroxidase 4, and system Xc-cystine/glutamate reverse transporter). Mechanistically, hUCMSC treatment inhibited c-Jun N-terminal kinase (JNK) and Kelch-like ECH-associated protein 1 (KEAP1) activation while increasing the expression of nuclear factor erythroid 2-related factor 2 (NRF2). Furthermore, pretreatment of HK-11 cells with NRF2 siRNA, the JNK inhibitor SP600125, or the JNK agonist anisomycin demonstrated the regulation of the JNK/KEAP1/NRF2 signaling pathway by hUCMSCs. Innovation and Conclusion: HUCMSCs inhibit ferroptosis in DN via the JNK/KEAP1/NRF2 signaling pathway, providing a new perspective and scientific evidence for treating DN. Antioxid. Redox Signal. 42, 807-826.

The effect and mechanism of albiflorin in alleviating hepatic fibrosis by mediating hepatocyte ferroptosis via the SLC7A11/GPX4 signaling pathway.

The cGAS/STING-mediated inflammatory response plays a key role in diabetic kidney disease (DKD) pathogenesis. However, effective anti-inflammatory therapies to halt disease its progression remain unavailable. The Qihuang Yishen (QHYS) formula has been demonstrated to reduce proteinuria and halt the course of DKD. However, the underlying mechanism of QHYS remains unclear. The purpose of this study was to determine whether QHYS had anti-inflammatory effects on DKD by targeting the cGAS/STING pathway both in vitro and in vivo. In vivo, DKD models were established in KK-Ay mice using a high-fat diet. In vitro, human renal tubular epithelial cells (HK-2) were exposed to high glucose (HG) and palmitic acid (PA) for 24 h to simulate renal tubular injury. Both experimental settings received interventions with QHYS or canagliflozin (positive control drug). Bioactive components in QHYS-containing serum were characterized by HPLC-ESI/MS. Body weight, random blood glucose, renal function markers, urinary protein levels, renal tubular injury indicators, and renal histological lesions were evaluated. Mitochondrial ultrastructure was analyzed by transmission electron microscopy (TEM). Western blotting quantified mitochondrial proteins TFAM and TOM20. Cell viability was assessed via CCK-8 assay, while ELISA kits measured IL-6 and TNF-α levels in cell supernatant. Quantitative PCR assessed expression of mtDNA and inflammatory mediators (IL-6, TNF-α, IFN-γ and CXCL10 mRNA). For cGAS/STING pathway analysis, key proteins were evaluated through western blotting, immunofluorescence, and immunohistochemistry. Additionally, mtDNA transfection experiments in HK-2 cells elucidated QHYS's regulatory mechanism on this pathway. HPLC-ESI/MS identified 32 principal bioactive components in QHYS. In vivo experiments showed that QHYS significantly reduced Scr and UREA levels (p < 0.05), decreased UACR, 24 h UTP and renal tubular injury markers NAG and NGAL levels (p < 0.01), and alleviated renal histopathological damage in KK-Ay mice, including glomerular hypertrophy, mesangial matrix expansion, tubular vacuolar degeneration, and tubulointerstitial inflammation. Moreover, QHYS ameliorated mitochondrial structural damage, upregulated the expression of TFAM and TOM20 (p < 0.05), and suppressed cGAS/STING pathway activation (p < 0.01). In vitro experiments demonstrated that QHYS alleviated mitochondrial damage (p < 0.05) and mtDNA leakage (p < 0.01), and inhibited cGAS/STING signaling and downstream inflammation (p < 0.05). More importantly, transfecting mtDNA into the cytoplasm to mimic the leakage process demonstrated that QHYS-containing serum directly inhibits cGAS/STING pathway activation (p < 0.05), and this mechanism also alleviated renal tubular injury in DKD. QHYS attenuates renal tubular inflammatory damage via cGAS/STING pathway regulation. This pioneering study provides the first evidence that herbal medicine exerts renal tubule protection through multi-target regulation of innate immune-mediated inflammatory injury in metabolic nephropathy, offering novel scientific evidence for DKD intervention.

Diabetic nephropathy (DN) is thought to be the major cause of end-stage renal disease. Due to its complicated pathogenesis and the low efficacy of DN treatment, a deep understanding of new etiological factors may be useful. Ferroptosis, a nonapoptotic form of cell death, is characterized by the accumulation of iron-dependent lipid peroxides to lethal levels. Ferroptosis-triggered renal tubular injury is reported to participate in the development of DN, and blocking ferroptosis might be an effective strategy to prevent the development of DN. Quercetin (QCT), a natural flavonoid that is present in a variety of fruits and vegetables, has been reported to ameliorate DN. However, its underlying nephroprotective mechanism is unclear. Herein, we explored the antiferroptosic effect of QCT and verified its nephroprotective effect using DN mice and high glucose (HG)-incubated renal tubular epithelial cell models. We found HG-induced abnormal activation of ferroptosis of renal tubular epithelial cells, and QCT treatment inhibited ferroptosis by downregulating the expression of transferrin receptor 1 (TFR-1) and upregulating the expression of glutathione peroxidase 4 (GPX4), ferritin heavy chain 1 (FTH-1), and the cystine/glutamate reverse antiporter solute carrier family 7 member (SLC7A11) in DN mice and HG-incubated HK-2 cells. Subsequently, both in vitro and in vivo results confirmed that QCT activated the NFE2-related factor 2 (Nrf2)/Heme oxygenase-1(HO-1) signaling pathway by increasing the levels of Nrf2 and HO-1. Therefore, this study supports that QCT inhibits the ferroptosis of renal tubular epithelial cells by regulating the Nrf2/HO-1 signaling pathway, providing a novel insight into the protective mechanism of QCT in DN treatment.

Glycosaminoglycans: use in treatment of diabetic nephropathy.

ObjectiveRenal fibrosis plays a pivotal role in the progression of renal dysfunction in diabetic kidney disease (DKD), with the macrophage-to-myofibroblast transition (MMT) serving as a central mechanism driving the advancement of renal fibrosis to end-stage renal disease (ESRD) in various chronic kidney conditions. Although the sodium-glucose cotransporter 2 (SGLT2) inhibitor dapagliflozin (DAPA) slows renal function decline in DKD, its antifibrotic mechanisms remain unclear. This study aimed to elucidate whether DAPA ameliorates renal fibrosis by suppressing MMT, and the underlying molecular mechanisms.MethodsWe established a DKD model in male C57BL/6J mice by inducing them with a high-fat diet (HFD) followed by streptozotocin (STZ) injection. The effects of DAPA on renal function parameters (serum creatinine (Scr), urinary albumin-to-creatinine ratio (UACR)), and renal pathological injury, fibrosis markers (α-smooth muscle actin (α-SMA), type I collagen (Col-I)) were comprehensively evaluated. Immunofluorescence (IF) and Western blot were employed to analyze MMT progression (F4/80+/α-SMA+ double-positive cells) and TGF-β1-Smad3/7 pathway activity.ResultsDAPA significantly reduced blood glucose levels, mitigated weight loss, and effectively inhibited type 2 diabetes-induced increase in Scr (43.78 ± 3.84 vs. 33.93 ± 6.77 µmol/L, P < 0.05) and UACR (89.17 ± 16.33 vs. 52.51 ± 10.51 mg/g, P < 0.05) in DKD mice. DAPA significantly attenuated glomerular hypertrophy, mesangial hyperplasia, and vacuolar degeneration of the tubular epithelium in DKD mice, while concurrently reducing glomerular and tubular injury scores, as well as the renal interstitial fibrosis area (P < 0.05). IF revealed increased MMT within the renal tubulointerstitium in DKD mice, accompanied by elevated deposition of α-SMA and Col-I. DAPA treatment markedly reduced F4/80+ macrophage infiltration and F4/80+/α-SMA+ and F4/80+/Col-I+ double-positive MMT cells, with decreased α-SMA and Col-I expression (P < 0.05). Additionally, TGF-β1 and phosphorylated Smad3 (p-Smad3) expression were significantly upregulated in DKD mice, whereas Smad7 was downregulated (P < 0.05). DAPA treatment significantly reduced TGF-β1 and p-Smad3 levels and restored Smad7 expression, rebalancing the Smad3/Smad7 axis (P < 0.05). Therefore, we propose that DAPA likely alleviates renal fibrosis in DKD by modulating renal TGF-β1 activity and restoring the Smad3/Smad7 balance, thereby suppressing MMT.ConclusionThis study is the first to reveal that DAPA is highly likely to exert anti-fibrotic effects through a novel mechanism involving targeted modulation of the TGF-β1-Smad3/7 axis to inhibit MMT, thereby laying a theoretical foundation and identifying a potential therapeutic target for precision treatment of DKD.

Renal tubular injury is a key factor in the progression of diabetic kidney disease to end-stage renal disease. Hyperoside, a natural flavonol glycoside in various plants, is a potentially effective drug for the clinical treatment of diabetic kidney disease. However, the specific mechanisms remain unknown. Therefore, this study will explore the effect and mechanism of hyperoside on renal tubulointerstitium in diabetic kidney disease. db/db mouse (C57BL/KsJ) is a model of type 2 diabetes resulting from Leptin receptor point mutations, with the appearance of diabetic kidney disease. Therefore, db/db mice were used for in vivo experimental studies. In vitro, human renal tubular epithelial cells were incubated with bovine serum albumin to simulate the injury of renal tubular epithelial cells caused by excessive albumin in primary urine. The experimental results showed that hyperoside could improve kidney function and reduce kidney tissue damage in mice, and could inhibit oxidative stress, extracellularly regulated protein kinases 1/2 signaling activation, and pyroptosis in human renal tubular epithelial cells. Therefore, hyperoside inhibited oxidative stress by regulating the activation of the extracellularly regulated protein kinases 1/2/mitogen-activated protein kinase signaling pathway, thereby alleviating proteinuria-induced pyroptosis in renal tubular epithelial cells. This study provides novel evidence that could facilitate the clinical application of hyperoside in diabetic kidney disease treatment.

Renal tubular interstitial injury plays a key role in the progression of diabetic nephropathy (DN) and, thus, the study of renal tubular injury in DN is important. The aim of the present study was to elucidate the role of the NLR family CARD domain containing 4 (NLRC4) inflammasome in renal tubular epithelial cell (RTEC) injury in DN. Human kidney biopsy tissues were obtained from patients with DN, and normal kidney tissues were obtained from nephrectomies performed for renal hamartoma. Human RTECs (HK2 cells) were divided into normal glucose (D-glucose 5.6 mmol/l), high glucose (HG; 30 mmol/l), high osmotic (D-glucose 5.6 mmol/l + D-mannitol 24.4 mmol/l), HG + NLRC4 small interfering (si)RNA or HG + siRNA control groups. Then, the expression levels of NLRC4, PTEN-induced kinase 1 (PINK1) and parkin, as well as the levels of mitochondrial reactive oxygen species, which are associated with mitophagy, were observed. The expression levels of NLRC4, PINK1, parkin and phosphorylated parkin in the RTECs of patients with DN were higher compared with those in normal controls. In HK2 cells, HG stimulated the expression of NLRC4, the secretion of IL-1β and IL-18 and cell death. Moreover, knockdown of NLRC4 expression in HK2 cells treated with HG reduced the secretion of the inflammatory cytokines, IL-1β and IL-18. The findings of the present study may provide a rationale for the development of treatments for patients with DN by preventing inflammasome activation.

Objective: To investigate the role of ferroptosis in renal cell injury induced by diquat (DQ) . Methods: From January to October 2022, human renal tubular epithelial (HK-2) cells were treated with DQ for 48 h, and different doses of ferroptosis inhibitors [deferoxamine (DFO), Fer-1] were added, and cells were harvested 24 h later. The experiment was divided into 6 groups (n=6) : control group, DQ group (60 μmol/L), 20 μmol/L DFO (DFO-H) group, 10 μmol/L DFO (DFO-L) group, 5 μmol/L Fer-1 (Fer-1-H) group, 0.5 μmol/L Fer-1 (Fer-1-L) group. From December 2022 to June 2023, male C57bl/6 mice were selected to establish the animal model, and the experimental group was divided into 4 groups (n=6) : control group, DQ group (25 mg/kg), DFO group (100 mg/kg) and Fer-1 group (2.5 μmol/kg). The changes of renal tissue were detected by HE staining. The fluorescence probe of ferrous ions was used to detect the change of iron ions in cells, and the colorimetric determination of total iron and ferrous ions in mouse kidney tissues was performed. Reverse transcription real-time quantitative polymerase chain reaction (qRT-PCR) and Western blotting were used to detect mRNA and protein expression changes related to ferroptosis signaling pathway. TUNEL staining was used to detect apoptosis. Enzyme-linked immunosorbent assay (ELISA) was used to detect the changes of antioxidant-related proteins and oxidative stress-related products. Differences among groups were analyzed by one-way analysis of variance. Results: In vitro test, compared with the control group, the iron ion level of HK-2 cells in DQ group was increased, the mRNA and protein expression levels of glutathione peroxidase 4 (GPX4), solute carrier family 7 member 11 (SLC7A11) and ferritin heavy chain (FTH) were decreased, the mRNA and protein expression levels of transferrin receptor 1 (TFR1) and divalent metal transporter 1 (DMT1) were increased, and the apoptosis level was significantly increased (P<0.05). The expression levels of glutathione (GSH) and super oxide dismutase (SOD) in HK-2 cells in DQ group were significantly lower than those in control group (P<0.05), and the expression levels of malondialdehyde (MDA) and reactive oxygen species (ROS) were significantly higher than those in control group (P<0.05). Compared with DQ group, iron ion levels in HK-2 cells in the intervention groups of DFO and Fer-1 at different doses were decreased (P<0.001), and GPX4, SLC7A11 and FTH mRNA and protein expression levels were increased, and the mRNA and protein expression levels of TFR1 and DMT1 were decreased in DFO-H and DFO-L groups (P<0.05). The apoptosis levels in the intervention groups of DFO and Fer-1 at different doses were decreased compared with DQ group (P<0.001), the expression levels of GSH and SOD were higher than those in DQ group (P<0.05), and the expression levels of ROS were lower than those in DQ group (P<0.05). In vivo, HE staining showed that the renal tissue of DQ group mice had obvious renal tubular epithelial cell injury with inflammatory cell infiltration. Compared with DQ group, DFO group and Fer-1 group had less damage of renal tubular epithelial cells and less inflammatory cell infiltration. Compared with the control group, the total iron content and ferrous iron content in kidney tissue of mice in DQ group were increased, the mRNA and protein expression levels of GPX4, SLC7A11 and FTH were decreased, the mRNA and protein expression levels of TFR1 and DMT1 were increased, and the apoptosis level was increased in DQ group (P<0.05). The levels of GSH and SOD in DQ group were lower than those in control group, while the levels of MDA and ROS in DQ group were higher than those in control group (P<0.05). Compared with DQ group, the total iron content and ferrous iron content in DFO group, and ferrous iron content in Fer-1 group were decreased (P<0.001), the mRNA and protein expression levels of GPX4, SLC7A11 and FTH in kidney tissues of mice in DFO group and Fer-1 group were increased (P<0.05), and the mRNA and protein expression levels of TFR1 and DMT1 were decreased (P<0.05). The level of apoptosis in DFO group and Fer-1 group was lower than that in DQ group (P<0.001). Compared with DQ group, the expression levels of GSH in kidney tissues, and the expression levels of SOD in serum and kidney tissues in DFO group were increased (P<0.05), and the expression levels of GSH and SOD in serum and kidney tissues in Fer-1 group were increased (P<0.05). The expression levels of MDA and ROS in serum and kidney tissues of DFO group and Fer-1 group were lower than those of DQ group (P<0.05) . Conclusion: Ferroptosis may be involved in renal cell injury induced by DQ poisoning, and ferroptosis inhibitor may alleviate DQ-induced renal injury by inhibiting ferroptosis.

Objective The long noncoding RNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) are closely associated with the pathogenesis of diabetic nephropathy (DN). But a complete mechanism for MALAT1 in DN has yet to be identified. This study investigated the effect of MALAT1 on DN through the regulation of miR-15b-5p/TLR4 signaling. Method Renal tissues were collected from DN patients. Human renal tubular epithelial cells (HK-2) were used as a model of DN induced by high glucose (HG). We then measured the viability, apoptosis, and inflammatory cytokine levels of HK-2 cells using the corresponding assays. Following transfections of si-MALAT1, si-MALAT1+miR-15b-5p inhibitor, or si-MALAT1+vector TLR4 into HG-stimulated HK-2 cells, cell viability, apoptosis, and inflammatory cytokines were again measured. Furthermore, dual-luciferase reporter assay validated the interactions of MALAT1/miR-15b-5p and miR-15b-5p/TLR4. In addition, the interaction between MALAT1 and miR-15b-5p was investigated by RNA immunoprecipitation (RIP). Results A significant upregulation of MALAT1 was observed in DN kidney tissues, as well as in HG-stimulated HK-2 cells. MALAT1 knockdown attenuates the inhibition of cell viability, apoptosis, and inflammatory response induced by HG in HK-2 cells. Moreover, a miR-15b-5p inhibitor or TLR4 overexpression reversed the above effects induced by MALAT1 knockdown. Conclusion These results indicate that reduced MALAT1 ameliorates HG-stimulated HK-2 cell damage through an inhibition of the miR-15b-5p/TLR4 axis. MALAT1 may serve as a biomarker and potential therapeutic target for DN.

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