Suppressing ferroptosis via modulating FTH1 by silybin for treatment of renal fibrosis.

Extracellular cold-inducible RNA-binding protein (eCIRP) was discovered as a potent damage-associated molecular pattern (DAMP). It has been shown that eCIRP is linked to various types of programmed cell death and acute inflammation. However, the role of eCIRP in chronic inflammation and renal fibrosis has not been elucidated. Accumulating evidence indicates that renal tubular epithelial cells (RTECs) play a significant role in renal fibrosis. C23, a small molecular peptide inhibitor of eCIRP, has been implicated as a therapeutic agent in the context of acute inflammation and tissue injury. PANoptosis or synchronized cell death is observed as simultaneous triggering of apoptosis, pyroptosis, and necroptosis. However, its role in renal fibrosis is not known. We therefore hypothesize that eCIRP induced-chronic inflammation and injury in RTECs are mediated by PANoptosis and that inhibition of eCIRP by C23 decreases RTEC PANoptosis and attenuates renal injury and fibrosis in a mouse model of unilateral ureter obstruction (UUO) injury. By using primary RTECs, we demonstrated that eCIRP induces inflammatory cytokines, Z-DNA-binding protein-1, and other PANoptosome markers and markers of apoptosis, pyroptosis, and necroptosis. We then substantiated that C23 downregulated proinflammatory cytokines and inhibited PANoptosis in the RTECs. Using the UUO mouse model, we demonstrated renal cell PANoptosis and renal fibrosis 7 days after UUO. Importantly, treatment with C23 effectively inhibited PANoptosis and concurrently ameliorated renal fibrosis. Taken together, eCIRP induces inflammation and PANoptosis in RTECs, whereas C23 inhibits PANoptosis in these cells and attenuates renal fibrosis in UUO mice.NEW & NOTEWORTHY Renal fibrosis is a common pathological manifestation of chronic kidney disease (CKD). Extracellular cold-inducible RNA-binding protein (eCIRP) was discovered as a potent damage-associated molecular pattern (DAMP). eCIRP is linked to various types of programmed cell death. PANoptosis or synchronized cell death is observed as simultaneous triggering of apoptosis, pyroptosis, and necroptosis. Inhibiting eCIRP by C23, a small molecular peptide inhibitor of eCIRP, attenuated PANoptosis and renal fibrosis in CKD.

Renal fibrosis is a critical pathogenesis of chronic kidney disease (CKD). Crocin, an active compound derived from saffron (Crocus sativus L.), exhibits potent antioxidant and renal protection properties. However, the effects of crocin on renal fibrosis and its underlying mechanisms remain unclear. This study aimed to investigate the potential role and molecular mechanism of crocin in protecting renal cells from ferroptosis and improving renal fibrosis. The study utilized a unilateral ureteral obstruction (UUO) rat model and erastin-induced ferroptosis model in rat renal tubular epithelial (NRK-52E) cells to evaluate the effect of crocin on ferroptosis and renal fibrosis. Phospho-specific antibody microarray analysis was conducted to profile crocin-relevant phosphate proteins among 16 signaling pathways. Surface plasmon resonance (SPR) assessments were employed to explore molecular interactions between crocin and mitogen-activated protein kinase kinase 4 (MKK4). Co-immunoprecipitation (Co-IP) experiments were used to investigate interactions between MKK4 and Nrf2. Crocin attenuated renal fibrosis as demonstrated by reducing profibrotic markers in both the UUO model and erastin-treated NRK-52E cells. It also significantly inhibited ferroptosis invivo and invitro. Microarray profiling and molecular docking suggested that MKK4 was a critical regulator of ferroptosis and fibrosis. SPR analysis indicated that crocin inhibited p-MKK4 by directly interacting with MKK4. Co-IP further revealed that p-MKK4 interacted with Nrf2, leading to alleviating reactive oxygen species (ROS) levels and subsequently attenuating renal ferroptosis. Crocin exerts protective effects against renal fibrosis and ferroptosis via modulation of phospho-MKK4/Nrf2/ferroptosis signaling axis. These findings suggest crocin is a promising renoprotective agent for renal fibrosis, and MKK4 represents a potential target for ferroptosis-related kidney injury.

ObjectiveRenal fibrosis is the most common manifestation of chronic kidney disease (CKD). Noting that existing treatments of renal fibrosis only slow disease progression but do not cure it, there is an urgent need to identify novel therapies. Hydrogen sulfide (H2S) is a newly discovered endogenous small gas signaling molecule exerting a wide range of biologic actions in our body. This review illustrates recent experimental findings on the mechanisms underlying the therapeutic effects of H2S against renal fibrosis and highlights its potential in future clinical application.Data sourcesLiterature was collected from PubMed until February 2019, using the search terms including “Hydrogen sulfide,” “Chronic kidney disease,” “Renal interstitial fibrosis,” “Kidney disease,” “Inflammation factor,” “Oxidative stress,” “Epithelial-to-mesenchymal transition,” “H2S donor,” “Hypertensive kidney dysfunction,” “Myofibroblasts,” “Vascular remodeling,” “transforming growth factor (TGF)-beta/Smads signaling,” and “Sulfate potassium channels.”Study selectionLiterature was mainly derived from English articles or articles that could be obtained with English abstracts. Article type was not limited. References were also identified from the bibliographies of identified articles and the authors’ files.ResultsThe experimental data confirmed that H2S is widely involved in various renal pathologies by suppressing inflammation and oxidative stress, inhibiting the activation of fibrosis-related cells and their cytokine expression, ameliorating vascular remodeling and high blood pressure, stimulating tubular cell regeneration, as well as reducing apoptosis, autophagy, and hypertrophy. Therefore, H2S represents an alternative or additional therapeutic approach for renal fibrosis.ConclusionsWe postulate that H2S may delay the occurrence and progress of renal fibrosis, thus protecting renal function. Further experiments are required to explore the precise role of H2S in renal fibrosis and its application in clinical treatment.

Background and Aims Renal fibrosis is the common pathological pathway of various chronic kidney diseases progressing to the end stage of renal failure. Anoctamin 3 (ANO3) has been identified as a K-channel regulator and been extensively studied in the field of neurobiology. The role of ANO3 in kidney diseases is currently unknown. Method Mouse models of renal fibrosis were established by unilateral ureteral obstruction (UUO) operation or aristolochic acid I (AAI) injection. Human proximal tubular cell line (HK2) was used as an in vitro model. ANO3 was deleted in vitro by siRNA. Western blotting analysis was performed. Glucogenic metabolites were measured. Results We found that ANO3 is upregulated in mouse kidneys with unilateral ureteral obstruction (UUO) or aristolochic acid nephropathy (AAN), and tightly correlated with the progression of renal fibrosis and negatively correlated with expression of several rate-limiting enzymes of renal gluconeogenesis. Knockdown of ANO3 with siRNA increased the concentration of glucose and reduced the concentration of lactate in the supernatant of TGF-β stimulated renal proximal tubular (HK2) cells. In parallel, transfection of ANO3 siRNA significantly reduced the expression of epithelial-to-mesenchymal transition (EMT) and fibrotic markers in TGF-β stimulated HK2 cells. We further showed that ANO3 expression is positively correlated with the expression of a methyltransferase enhancer of zeste homologue 2 (EZH2) in UUO and AAN mouse kidneys by time. Transfection of ANO3 siRNA significantly reduced the expression of EZH2 in TGF-β stimulated HK2 cells. Overexpression of EZH2 by adenovirus reversed the pro-gluconeogenic and anti-fibrotic effect of ANO3 siRNA in TGF-β stimulated HK2 cells by measuring the concentration of glucose and lactate in supernatant and analyzing the expression of EMT or fibrotic markers. Conclusion In conclusion, ANO3 inhibits renal gluconeogenesis through EZH2 and contributes to renal fibrosis. ANO3 could be promising therapeutic target to inhibit renal fibrosis in chronic kidney disease patients.

BackgroundRenal fibrosis represents the final common pathological manifestation of chronic kidney disease (CKD), yet the underlying mechanism remains elusive, and there is still a lack of effective targeted therapeutic strategy. Although previous research indicated that repressor element 1-silencing transcription factor (REST) contributed to acute kidney injury (AKI) in renal tubular epithelial cells (RTECs), its specific contribution to renal fibrosis and associated mechanisms remains largely unexplored.MethodsRenal biopsies from CKD patients were collected to evaluate the expression of REST. Kidney-specific Rest conditional knockout (Cdh16-Cre/Restflox/flox) mice were generated and employed unilateral ureter obstruction (UUO) models to investigate the role of REST in renal fibrosis. RNA sequencing was performed to elucidate the mechanism. Mitochondrial function was evaluated by transmission electron microscopy (TEM), reactive oxygen species (ROS), oxygen consumption rates (OCR), extracellular acidifcation rate (ECAR) and adenosine triphosphate (ATP). The severity of renal fibrosis was assessed through Western blot, immunofluorescent staining and immumohistochemical staining. Bioinformatic prediction, dual luciferase reporter gene assay, point mutation and chromatin immunoprecipitation (ChIP) assay were utilized to clarify the molecular mechanism.ResultsREST was significantly up-regulated in the kidney tissues from CKD patients, UUO-induced fibrotic mouse models and TGF-β1-incubated RTECs. Notably, kidney-specific knockout of Rest prominently alleviated renal fibrosis by improving mitochondrial energy metabolism and restoring fatty acid oxidation. Mechanically, REST disturbed mitochondrial energy metabolism through repressing the transcription of oxoglutarate dehydrogenase-like (OGDHL) via directly binding to its promotor region. Further, pharmacological inhibition of REST using the specific REST inhibitor, X5050, significantly ameliorated the progression of renal fibrosis both in vitro and in vivo.ConclusionsOur explorations revealed the upregulation of REST in renal fibrosis disrupts mitochondrial energy metabolism through transcriptionally suppressing OGDHL, which may act as a promising therapeutic target for renal fibrosis.

Renal fibrosis is the final manifestation of chronic kidney disease (CKD) regardless of etiology. Hypoxia-inducible factor-2 alpha (HIF-2α) is an important regulator of chronic hypoxia, and the late-stage renal tubular HIF-2α activation exerts protective effects against renal fibrosis. However, its specific role in progressive renal fibrosis remains unclear. Here, we investigated the effects of the long-term tubular activation of HIF-2α on renal function and fibrosis, using in vivo and in vitro models of renal fibrosis. Progressive renal fibrosis was induced in renal tubular epithelial cells (TECs) of tetracycline-controlled HIF-2α transgenic (Tg) mice and wild-type (WT) controls through a 6-week adenine diet. Tg mice were maintained on doxycycline (DOX) for the diet period to induce Tg HIF-2α expression. Primary TECs isolated from Tg mice were treated with DOX (5 μg/ml), transforming growth factor-β1 (TGF-β1) (10 ng/ml), and a combination of both for 24, 48, and 72 hr. Blood was collected to analyze creatinine (Cr) and blood urea nitrogen (BUN) levels. Pathological changes in the kidney tissues were observed using hematoxylin and eosin, Masson's trichrome, and Sirius Red staining. Meanwhile, the expression of fibronectin, E-cadherin and α-smooth muscle actin (α-SMA) and the phosphorylation of p38 mitogenactivated protein kinase (MAPK) was observed using western blotting. Our data showed that serum Cr and BUN levels were significantly lower in Tg mice than in WT mice following the adenine diet. Moreover, the protein levels of fibronectin and E-cadherin and the phosphorylation of p38 MAPK were markedly reduced in the kidneys of adenine-fed Tg mice. These results were accompanied by attenuated fibrosis in Tg mice following adenine administration. Consistent with these findings, HIF-2α overexpression significantly decreased the expression of fibronectin in TECs, whereas an increase in α-SMA protein levels was observed after TGF-β1 stimulation for 72 hr. Taken together, these results indicate that long-term HIF-2α activation in CKD may inhibit the progression of renal fibrosis and improve renal function, suggesting that long-term renal HIF-2α activation may be used as a novel therapeutic strategy for the treatment of CKD. [BMB Reports 2023; 56(3): 196-201].

Renal fibrosis, a terminal manifestation of chronic kidney disease, is characterized by uncontrolled inflammatory responses, increased oxidative stress, tubular cell death, and imbalanced deposition of extracellular matrix. 5,2'-Dibromo-2,4',5'-trihydroxydiphenylmethanone (LM49), a polyphenol derivative synthesized by our group with excellent anti-inflammatory pharmacological properties, has been identified as a small-molecule inducer of extracellular matrix degradation. Nonetheless, the protective effects and mechanisms of LM49 on renal fibrosis remain unknown. Here, we report LM49 could effectively alleviate renal fibrosis and improve filtration function. Furthermore, LM49 significantly inhibited macrophage infiltration, pro-inflammatory cytokine production and oxidative stress. Interestingly, in HK-2 cells induced by tumour necrosis factor alpha under oxygen-glucose-serum deprivation conditions, LM49 treatment similarly yielded a reduced inflammatory response, elevated cellular viability and suppressed cell necrosis and epithelial-to-mesenchymal transition. Notably, LM49 prominently suppressed the high-mobility group box 1 (HMGB1) expression, nucleocytoplasmic translocation and activation. Mechanistically, drug affinity responsive target stability and cellular thermal shift assay confirmed that LM49 could interact with the target heat shock protein 90 alpha family class A member 1 (Hsp90α), disrupting the direct binding of Hsp90α to HMGB1 and inhibiting the nuclear export of HMGB1, thereby suppressing the inflammatory response, cell necrosis and fibrogenesis. Furthermore, molecular docking and molecular dynamic simulation revealed that LM49 occupied the N-terminal ATP pocket of Hsp90α. Collectively, our findings show that LM49 treatment can ameliorate renal fibrosis through inhibition of HMGB1-mediated inflammation and necrosis via binding to Hsp90α, providing strong evidence for its anti-inflammatory and anti-fibrotic actions.

Background and Aims Diabetic nephrology (DN) is one of the most common and devastating complications of diabetic mellitus, making the leading cause of end stage renal disease (ESRD) all over the world. And there is no satisfactory therapy to delay DN progression in clinic. Renal tubular epithelial cell (RTEC) senescence is an important biological event in the progression of DN. Decoy receptor 2 (DcR2), a transmembrane receptor for TRAIL, is specifically highly expressed in the membrane and cytoplasm of senescent RTECs, and promotes renal fibrosis in DN. This study is aim to design DcR2 antagonistic peptide, and confirm its role and mechanism in alleviating renal fibrosis in DN. Method A phage display peptide library was employed to screen candidate DcR2 antagonistic peptides after three rounds of elution and neutralization through 12 peptide libraries, based on DcR2 protein. The best DcR2 antagonistic peptide was selected according to mass spectrometry, HPLC detection and affinity test by bio-layer interferometry (BLI). This study was conducted by setting high glucose (HG) models based on primary RTECs in vitro. Firstly, CCK8 was applied to measure the effect of DcR2 antagonistic peptide on cell viability. Immunofluorescence staining was used to confirm the colocalization relationship between DcR2 and its antagonistic peptide. SA-β-gal staining showed the influence of DcR2 antagonistic peptide on RTEC senescence. Then we assessed senescent biomarkers like p21, p16 and DcR2 by quantitative PCR (qPCR). Western blot (WB) was used to evaluate the effect of DcR2 antagonistic peptide on cellular senescence and renal fibrosis by senescent biomarker p21, p16, DcR2, γ-H2AX, LaminB1, etc and fibrotic marker like CollagenⅠ.Finally, Immunofluorescence staining of α-SMA, a fibrotic marker, also measured its impact on renal fibrosis. Results Ten candidate DcR2 antagonistic peptides were screened by a phage display peptide library. And the best DcR2 antagonistic peptide, namely RF8 was selected with purity of 91.59% and dissociation constant of 9.76E−05 M. CCK8 showed that RF8 enhanced cell viability in a concentration-dependent manner. The colocalization assay exhibited favorable combination of DcR2 and RF8 in vitro. SA-β-gal staining demonstrated RF8 largely reduced the expression of senescent RTECs in HG group. After administration of RF8, the mRNA levels of p16 and p21 were significantly inhibited. WB indicated RF8 reduced the expression of senescent and fibrotic markers induced by HG. Similarly, immunofluorescence staining demonstrated that HG-induced increase of α-SMA was negated by RF8, which indicated well inhibition of RF8 on RTEC senescence and renal fibrosis in vitro. Conclusion This study reveals that RF8 is a potent therapeutic remedy that mitigate RTEC senescence and renal fibrosis, which could relieve the progression of DN by targeted antagonism of DcR2, providing hope for the treatment of DN.

The antifibrotic and anti-inflammatory effects of icariin on the kidney in a unilateral ureteral obstruction mouse model

Background and Aims Cell senescence of renal tubular epithelial cells (RTECs), which is involved in renal tubulointerstitial fibrosis (TIF), is a key event in the progression of diabetic nephropathy (DN). However, the underlying mechanism remains unclear. This study aims to investigate the role and mechanism of decoy receptor 2 (DcR2) in TIF and cell senescence of RTECs. Method Streptozotocin (STZ)-induced DN mice model and high glucose (HG)-induced cell senescent model were constructed. The expression of DcR2 were regulated by ultrasound-mediated gene transfer of DcR2-siRNA or DcR2-overexpression plasmid in vivo. The co-expression of DcR2 with senescent markers (p16, p21 and SA-β-gal) and fibrotic markers (FN, collagen I and α-SMA). The interaction of DcR2 and its ligand TRAIL and antagonistic receptor DR5 were detected by laser confocal and Co-IP. The DcR2-interaction proteins were screened in renal tissue of DN patients and HG-induced cells by Co-IP combining with LC-MS/MS. The interaction of DcR2 and PRDX1 was validated by Co-IP, pull down assay and immunofluorescence co-staining. Peroxidase activity of PRDX1 was assessed by the kits of reactive oxygen species (ROS) and specific 2-cys peroxidase activity. The level of PRDX1 phosphorylation was measured through WB and the phosphorylated site were studied by wild-type PRDX1 and mutant Tyr194 PRDX1. Results DcR2 was co-expressed with senescent markers and co-localization with fibrotic markers in DN patients and STZ-DN mice. Knockdown of DcR2 effectively decreased renal fibrosis and tubular cell senescence in STZ-induced DN mice. However, DcR2 overexpression showed the opposite effects. DcR2 didn’t interacted with TRAIL or DR5 in vivo and vitro. The following study indicated that PRDX1 is a novel DcR2-interacting protein using quantitative proteomics, which have peroxidase and antioxidant activity in cytoplasm. The interaction of DcR2-PRDX1 mediated RTEC senescence in vitro. Knockdown of DcR2 inhibited the level of ROS and promoted the peroxidase activity of PRDX1, resulting in oxidative stress and cell senescence, and DcR2 overexpression presented the contrary results. DcR2 regulated the level of PRDX1 phosphorylation by interacting Tyr194 of PRDX1, and the mutant Tyr194 of PRDX1 can’t be phosphorylated by DcR2. Conclusion DcR2 mediates renal fibrosis and RTEC senescence by phosphorylating with PRDX1, suggesting DcR2 could serve as a potential therapeutic target for the amelioration of DN progression.

CXCR4 inhibition attenuates calcium oxalate crystal deposition-induced renal fibrosis

Chronic hyperglycemia induces intrarenal oxidative stress due to the excessive production of reactive oxygen species (ROS), leading to a cascade of events that contribute to the development and progression of diabetic kidney disease (DKD). NOX5, a pro-oxidant NADPH oxidase isoform, has been identified as a significant contributor to renal ROS in humans. Elevated levels of renal ROS contribute to endothelial cell dysfunction and associated inflammation, causing increased endothelial permeability, which can disrupt the renal ecosystem, leading to progressive albuminuria and renal fibrosis in DKD. This study specifically examines the contribution of endothelial cell-specific human NOX5 expression in renal pathology in a transgenic mouse model of DKD. This study additionally compares NOX5 with the previously characterized NADPH oxidase, NOX4, in terms of their relative roles in DKD. Regardless of NOX4 pathway, this study found that endothelial cell-specific expression of NOX5 exacerbates renal injury, albuminuria and fibrosis. This is attributed to the activation of the endothelial mesenchymal transition (EMT) pathway via enhanced ROS formation and the modulation of redox-sensitive factors. These findings underscore the potential therapeutic significance of NOX5 inhibition in human DKD. The study proposes that inhibiting NOX5 could be a promising approach for mitigating the progression of DKD and strengthens the case for the development of NOX5-specific inhibitors as a potential therapeutic intervention.

Mitochondria were first described in 1840 as bioblasts, elementary organisms responsible for vital cellular functions, but were subsequently named mitochondria, from the Greek names mitos (thread) and chondros (granule), which describes their appearance during spermatogenesis.1 Their discovery generated substantial interest given their structure resembling bacteria, which led in subsequent years to important scientific discoveries positioning mitochondria as the energy powerhouse of the cell. The unique architecture of mitochondria, consisting of 2 membranes (outer and inner) and compartments (intermembrane space and matrix), is crucial for their vital functions. Mitochondria serve not only as primary sources of cellular energy, but also modulate several cellular processes, including oxidative phosphorylation, calcium homeostasis, thermogenesis, oxygen sensing, proliferation, and apoptosis.2 Therefore, mitochondrial injury and dysfunction might be implicated in the pathogenesis of several diseases. Hypertension accounts for nearly 30% of patients reaching end-stage renal disease.3 Renal injury secondary to hypertension or to ischemia associated with renovascular hypertension (distal to renal artery stenosis) may have significant and detrimental effect on health outcomes. Studies have highlighted several deleterious pathways, including inflammation, oxidative stress, and fibrosis that are activated in the hypertensive kidney, eliciting functional decline.4,5 However, the precise molecular mechanisms responsible for renal injury have not been fully elucidated. Over the past few years, increasing evidence has established the experimental foundations linking mitochondrial alterations to hypertensive renal injury (Table). Mitochondriopathies, abnormalities of energy metabolism secondary to sporadic or inherited mutations in nuclear or mitochondrial DNA (mtDNA) genes, may contribute to the development and progression of hypertension and its complications. In addition, several studies have reported mitochondrial damage and dysfunction consequent to hypertensive renal disease. View this table: Table. Evidence of Renal Mitochondrial Damage in Models of Hypertension and Antihypertensive Treatment Importantly, hypertensive-induced renal injury is characterized by activation of several deleterious pathways, including oxidative stress, renin–angiotensin–aldosterone …

Paricalcitol attenuates cyclosporine-induced kidney injury in rats

Diosmin attenuates UUO-induced renal ferroptosis and fibrosis by inhibiting the HIF-1α/FABP4 signaling axis.