Renal Fibrosis In Mice Research Articles

Deubiquitinase inhibitor bAP-15 suppresses renal epithelial to mesenchymal transition via inhibition of p300 stability

Renal fibrosis is an irreversible disease that is common in patients with chronic kidney disease. Elevated levels of the histone acetyltransferase p300 have been reported in various fibrotic diseases, including renal fibrosis, suggesting that p300 may be a promising therapeutic target. To investigate the specific deubiquitinase (DUB) involved in the regulation of p300 protein stability in renal epithelial cells, we tested 13 DUB inhibitors using a kidney tubular epithelial cell line. We found that the p300-specific DUB inhibitor, bAP-15 reduces p300 protein stability by targeting ubiquitin-specific protease 14 (USP14) and ubiquitin C-terminal hydrolase L5 (UCHL5). The mRNA levels of USP14 and UCHL5 were increased in patients with chronic kidney disease, and increased protein levels of USP14 and UCHL5 during fibrosis progression were validated using a mouse renal fibrosis model. Both USP14 and UCHL5 interacted with p300 in kidney tubular epithelial cells, with increased binding affinity in response to TGF-β. Moreover, bAP-15-induced p300 degradation inhibited epithelial-to-mesenchymal transition and reduced the expression of pro-fibrotic target genes. Our findings demonstrate an anti-fibrotic effect of bAP-15 through the regulation of p300 stability and suggest that bAP-15 may be a potential therapeutic agent for renal fibrosis.

Emodin Inhibits AIM2 Inflammasome Activation via Modulating K27-Linked Polyubiquitination to Attenuate Renal Fibrosis.

Chronic kidney diseases (CKD) is a serious threat to people's health with renal fibrosis as the major pathological feature. The absent in melanoma 2 (AIM2) has recently been proposed to play a critical role in CKD. Emodin is a major bioactive compound from rhubarb, which is widely used for clinical treatment of renal disease. The aim of this study is to elucidate the effect of emodin on unilateral ureteral obstruction (UUO) model mice and its association with the AIM2 inflammasome. In this study, we established the UUO-induced mice renal interstitial fibrosis invivo and bone marrow-derived macrophages (BMDMs) model invitro. The BUN, SCr, TNF-α, IL-1β in serum were examined. The degree of renal damage and fibrosis were determined by histological assessment. Immunofluorescence, western blot, and Co-IP were used to determine the mechanisms of emodin against CKD. Emodin could improve UUO-induced abnormal renal function and histopathological abnormalities. It could also ameliorate renal fibrosis, evidenced by inhibiting the expression of α-SMA, TGF-β1, FN, and collagen I. Mechanistically, emodin significantly suppressed AIM2 inflammasome as well as its components including ASC, cleaved caspase-1, and IL-1β both invivo and invitro. Further studies demonstrated that emodin inhibited K27-linked polyubiquitination of AIM2 by targeting on K64 sites of the lysine residues. In summary, emodin could hinder the activation of AIM2 inflammasome in UUO model mice through K27-linked polyubiquitination to reduce renal fibrosis. Emodin is a possible therapeutic option for CKD treatment.

Multi-omics analysis reveals the protective effects of Chinese yam polysaccharide against cisplatin-induced renal interstitial fibrosis

BackgroundChinese yam polysaccharide (SYDT) has been reported to protect renal function and mitigate renal fibrosis in mice with diabetic nephropathy. Based on a multi-omics analysis, the objectives of this study were to determine the effect of SYDT on cisplatin (CDDP)-induced chronic renal interstitial fibrosis (RIF) and the underlying molecular mechanisms using an in vivo model. MethodsRats were intraperitoneally injected with a single dose of CDDP and then treated with SYDT or amifostine (AMF). The levels of urinary N-acetyl-β-D-glucosaminidase (NAG), blood urea nitrogen (BUN) and serum creatinine (Scr) were detected to assess renal function. Renal tissue damage and fibrosis were evaluated using hematoxylin and eosin (H&E) and Masson's trichrome staining, respectively. In addition, this study applied transcriptomics and metabolomics to predict the possible mechanism of SYDT action, which was verified by several relevant examinations. ResultsSYDT significantly protected the renal function, alleviated renal tissue damage and fibrosis, as well as decreased the protein levels of vimentin, α-SMA and CTGF, whereas SYDT significantly increased MMP-1 protein level in renal tissues from rats treated with CDDP. There were 1130 differently expressed genes (DEGs) between the CDDP model and SYDT-M groups proved by transcriptome analysis, indicating that metabolic pathways were likely the primary targets of relevance. Consistent with the transcriptome analysis, metabolome analysis identified 276 differentially expressed metabolites (DEMs) between the SYDT-M and CDDP model groups, with predominant clustering within glycerophospholipid metabolism. Integrative analysis of the transcriptome and metabolome indicated that SYDT inhibited the glycerophospholipid metabolism pathway by regulating the target genes Gpd2, Gpam, Agpat3, Lcat, and Pla2g4b. Notably, integrative analysis showed that the Phospholipase D (PLD) signaling pathway may be the most relevant target. Moreover, related signaling pathway analysis confirmed that SYDT inhibited CDDP-induced RIF in rats by down-regulating the PLD pathway. ConclusionOur study showed that the alleviation of CDDP-induced RIF in vivo can be achieved through the inhibition of glycerophospholipid metabolism and PLD signaling pathways by SYDT.

Salvianolic acid B improves DEN/CCl4/C2H5OH-induced renal fibrosis by regulating YAP/TAZ and pSmad3C/pSmad3L

Salvianolic acid B (Sal B) is a natural compound extracted from the root of Salvia miltiorrhiza, which has multiple biological activities. Targeting Hippo pathway transcriptional coactivators YAP/TAZ combined with Smad3 is an effective strategy against renal fibrosis. However, whether Sal B improves DEN/CCl4/C2H5OH-induced renal fibrosis and regulates YAP/TAZ and Smad3 phospho-isoform, pSmad3C/pSmad3L is unknown. Presently, we used DEN/CCl4/C2H5OH to induce renal fibrosis in mice and TGF-β1 or MST1/2 inhibitor XMU-MP-1 to induce HK-2 cells to investigate the activity and molecular mechanisms of Sal B. The results demonstrated that Sal B ameliorated DEN/CCl4/C2H5OH-induced renal fibrosis, including reducing renal tissue injury, improving renal function, and decreasing collagen synthesis. Furthermore, Sal B downregulated YAP/TAZ and promoted their phosphorylation in vivo and in vitro. Moreover, Sal B upregulated pSmad3C but downregulated pSmad3L in vivo and in vitro. Collectively, this study revealed that Sal B may improve DEN/CCl4/C2H5OH-induced renal fibrosis by regulating YAP/TAZ and pSmad3C/pSmad3L.

Qingshen granules inhibits dendritic cell glycolipid metabolism to alleviate renal fibrosis via PI3K-AKT-mTOR pathway

BackgroundQingshen exhibits anti-inflammatory and immunoregulation effects to renal damage. Dendritic cells (DCs) play a critical role in regulating the pathologic inflammatory environment in renal fibrosis (RF). PurposeTo investigate the immune modulation mechanism of qingshen granule (QSG) in RF, particularly focusing on the role of DCs. Methods/Study designAdenine-induced RF animal models were used to study the pharmacological effects of QSG and the immune cells differentiation and function. Glucose uptake, non-esterified fatty acids secretion, mitochondrial membrane potential (MMP) detection, and qPCR were used to explore the effect of QSG to glucose and lipid metabolism in DCs and T cells. The effect of QSG to PI3K-AKT-mTOR axis and the modulation of mTOR to PD-L1 were explored by co-culture experiments, co-immunoprecipitation and western blot assays. The interaction of DCs/CD8+T cells and renal tubular epithelial cells (RTECs) was investigated to demonstrate the direct action and/or the immune-mediated regulation of QSG to RF. The components of QSG in the serum were determined by HPLC. And the effect of active ingredients and formula to DCs and T cells was analyzed by cell experiments in vitro. ResultsQSG reduced nephritic histopathological damage and suppressed the release of proinflammatory cytokines in adenine-induced RF mice. Of note, QSG decreased the levels of CD86, MHC-II, and CCR7 on DCs, while, increased PD-L1 expression on DCs in RF. The results demonstrated that QSG promoted the maturation and inhibited the migration of DCs, and QSG decreased the antigen presenting of DCs to T cells. Additionally, QSG reduced the MMP and glucose/lipid utilization ratio in DCs. QSG also down-regulated the level of targeted metabolic genes included glucose transporter 1 (Glut1), sterol-regulatory element-binding protein 1 (Srebp1), acetyl-CoA carboxylase alpha (Acaca), phosphomevalonate kinase (Pmvk), and up-regulated sirtuin2 (Sirt2) in DCs. In terms of mechanism, QSG inhibited the metabolism-related PI3K-AKT-mTOR pathway, followed by regulating the interaction of mTOR with PD-L1 to enhance the membrane stability of PD-L1. Besides, HPLC analysis identified five active ingredients in QSG. The specific anti-inflammatory and immunosuppressive actions of these ingredients were found to be weaker than QSG as a whole. Finally, inhibiting DC function by QSG disrupted the communication among DCs, T cells, and RTECs. This disruption was associated with low expression of α-smooth muscle actin (α-SMA) and collagen type I (Col-I) in the kidney. ConclusionsQSG inhibits DC metabolism and function via the PI3K-AKT-mTOR pathway to alleviate RF. The study highlights the importance of the specific composition of the formula in targeting DC-mediated immune regulation.

Attenuation of renal fibrosis in mice due to lack of bombesin receptor-activated protein homologue.

Bombesin receptor-activated protein (BRAP), encoded by the C6orf89 gene in humans, is expressed in various cells with undefined functions. BC004004, the mouse homologue of C6orf89, has been shown to play a role in bleomycin-induced pulmonary fibrosis through the use of a BC004004 gene knockout mouse (BC004004-/-). In this study, we investigated the potential involvement of BRAP in renal fibrosis using two mouse models: unilateral ureteral obstruction (UUO) and type 2 diabetes mellitus induced by combination of a high-fat diet (HFD) and streptozocin (STZ). BRAP or its homologue was expressed in tubular epithelial cells (TECs) in the kidneys of patients with chronic kidney disease (CKD) and in BC004004+/+ mice. Compared to control mice, BC004004-/- mice exhibited attenuated renal injury and renal fibrosis after UUO or after HFD/STZ treatment. Immunohistochemistry and immunoblot analyses of the kidneys of BC004004+/+ mice after UUO surgery showed a more significant decrease in E-cadherin expression and a more significant increase in both α smooth muscle actin (α-SMA) and vimentin expression compared to BC004004-/- mice. Additionally, stimulation with transforming growth factor-β1 (TGF-β1) led to a more significant decrease in E-cadherin expression and a more significant increase in α-SMA and vimentin expression in isolated TECs from BC004004+/+ than in those from BC004004-/- mice. These results suggest that an enhanced epithelial-mesenchymal transition (EMT) process occurred in TECs in BC004004+/+ mice during renal injury, which might contribute to renal fibrosis. The loss of the BRAP homologue in BC004004-/- mice suppressed EMT activation in kidneys and contributed to the suppression of fibrosis during renal injury.

Polysaccharides from Sacha Inchi shell reduces renal fibrosis in mice by modulating the TGF-β1/Smad pathway and intestinal microbiota

Renal fibrosis is a common pathway involved in the progression of various chronic kidney to end-stage diseases, posing a substantial global public health challenge in the search for effective and safe treatments. This study investigated the effects and mechanisms of sacha inchi shell polysaccharide (SISP) on renal fibrosis induced by a high-salt diet (HSD) in mice. By analysing kidney-related protein pathways and the structure of gut microbiota, we found that SISP significantly reduced urinary protein levels induced by a HSD from 41.08 to 22.95 μg/mL and increased urinary creatinine from 787.43 to 1294.50 μmol/L. It reduced renal interstitial collagen fibres by 11.30 %, thereby improving the kidney function. SISP lowered the mRNA expression of TGF-B1, fibronectin, α-SMA, Smad2/3, and TGFBRII, leading to decreased protein levels of TGF-β1, p-Smad2/3, p-TGFβRII, fibronectin, α-SMA, p-Smad2/3/Smad2/3, and p-TGFβRII/TGFβRII. These changes blocked downstream transcription in the TGF-β1/Smad signalling pathway, thereby attenuating renal fibrosis in HSD mice. In addition, SISP altered the intestinal flora imbalance in HSD mice by reducing the relative abundance of the genera, Akkermansia, Faecalibaculum, and unidentified_Ruminococcaceae, and reversing the decline in the levels of the genera, Lactobacillus and Bacteroides. In conclusion, SISP is a promising nutraceutical for renal fibrosis management.

Evaluation of the novel ALK5 inhibitor EW-7197 on therapeutic efficacy in renal fibrosis using a three-dimensional chip model.

EW-7197, a potent oral ALK5 inhibitor, was assessed for its impact on transforming growth factor beta 1 (TGF-β1)-induced fibrosis in a three-dimensional (3D) renal fibrosis-on-a-chip and a mouse model. The evaluation included tubular epithelial-mesenchymal transition, angiogenesis, and inflammatory cytokine expression. In a 3D renal fibrosis-on-a-chip model, three cell types (kidney fibroblasts, human proximal tubular cell line, and human umbilical vein endothelial cells) were cultured and treated with TGF-β1 and EW-7197. Expression of alpha smooth muscle actin (α-SMA) and keratin 8 (KRT-8) was assessed, angiogenesis was observed via confocal microscopy, and cytokine levels were measured using real-time polymerase chain reaction, immunoassay, and enzyme-linked immunosorbent assay. In a cisplatin-induced renal fibrosis mouse model, blood urea nitrogen levels, TGF-β, and Smad 2/3 were determined, and renal fibrosis was assessed with Masson's trichrome stain. The α-SMA expression was significantly lower in the EW-7197 group than in the TGF-β fibrosis group. TGF-β decreased the expression of the epithelial marker KRT-8, an effect that was reversed by EW-7197 and SB431542. In the TGF-β-induced fibrosis model, the length of the thick vessels was reduced, and the diameter of both thick and thin vessels was decreased, but EW-7197 reversed these effects. EW-7197 significantly reduced the messenger RNA expression of TGF-β and increased the levels of vascular endothelial growth factor receptor 2, interleukin (IL)-10, and IL-6. EW-7197 reduced the levels of secretory cytokines TGF-β1, TGF-β3, IL-1β. In the cisplatin-induced renal fibrosis mouse model, EW-7197 reduced renal fibrosis by down-regulating TGF-β signaling. EW-7197 attenuated the TGF-β1-induced fibrotic cellular response in the 3D chip model and animal model. These findings indicate the potential effect of EW-7197 in attenuating renal fibrosis.

Deficiency of thiosulfate sulfurtransferase mediates the dysfunction of renal tubular mitochondrial fatty acid oxidation in diabetic kidney disease.

One of the main characteristics of diabetic kidney disease (DKD) is abnormal renal tubular fatty acid metabolism, especially defective fatty acid oxidation (FAO), accelerating tubular injury and tubulointerstitial fibrosis. Thiosulfate sulfurtransferase (TST), a mitochondrial enzyme essential for sulfur transfer, is reduced in metabolic diseases like diabetes and obesity. However, the potential role of TST in regulating fatty acid metabolic abnormalities in DKD remains unclear. Here, our data revealed decreased TST expression in the renal cortex of DKD patients. TST deficiency exacerbated tubular impairment in both diabetic and renal fibrosis mouse models, while sodium thiosulfate treatment or TST overexpression mitigated renal tubular injury with high-glucose exposure. TST downregulation mediated the decrease in S-sulfhydration of very long-chain specific acyl-CoA dehydrogenase, resulting in mitochondrial FAO dysfunction. This sequence of events exacerbates the progression of tubulointerstitial injury in DKD. Together, our findings demonstrate TST as a regulator of renal tubular injury in DKD.

Qingshen Granules alleviates renal fibrosis in mice by regulating exosomes, miR-330-3p, and CREBBP expression

To explore the effects of Qingshen Granules (QSG) on adenine-induced renal fibrosis in mice and in uric acid (UA)-stimulated NRK-49F cells and its mechanism for regulating exosomes, miR-330-3p and CREBBP. A mouse model of adenine-induced renal fibrosis were treated daily with QSG at 8.0 g·kg-1·d-1 via gavage for 12 weeks. An adenoassociated virus vector was injected into the tail vein, and renal tissues of the mice were collected for analyzing exosomal marker proteins CD9, Hsp70, and TSG101 and expressions of Col-III, α-SMA, FN, and E-cad using Western blotting and immunofluorescence and for observing pathological changes using HE and Masson staining. In the cell experiment, NRK-49F cells were stimulated with uric acid (400 μmol/L) followed by treatment with QSG-medicated serum from SD rats, and the changes in expressions of the exosomal markers and Col-III, α-SMA, FN, and E-cad were analyzed. Dual luciferase reporter assay was employed to examine the targeting relationship between miR-330-3p and CREBBP, whose expressions were detected by RT-qPCR and Western blotting in treated NRK-49F cells. The mouse models of adenine-induced renal fibrosis showed significantly increased levels of CD9, Hsp70, and TSG101, which were decreased by treatment with QSG. The expressions of Col-III, α-SMA, and FN increased and Ecad decreased in the mouse models but these changes were reversed by QSG treatment. QSG treatment obviously alleviated renal fibrosis in the mouse models. Intravenous injection of adeno-associated viral vector obviously inhibited miR-330-3p, increased CREBBP levels, and reduced fibrosis in the mouse models. Dual luciferase assay confirmed CREBBP as a target of miR-330-3p, which was consistent with the results of the cell experiments. QSG inhibits renal fibrosis in mice by regulating the exosomes, reducing miR-330-3p levels, and increasing CREBBP expression.

Astragalus mongholicus bunge and panax notoginseng formula (A&P) improves renal fibrosis in UUO mice via inhibiting the long non-coding RNA A330074K22Rik and downregulating ferroptosis signaling

BackgroundChronic kidney disease (CKD) and its associated end-stage renal disease (ESRD) are significant health problems that pose a threat to human well-being. Renal fibrosis is a common feature and ultimate pathological outcome of various CKD leading to ESRD. The Astragalus mongholicus Bunge and Panax notoginseng formula (A&P) is a refined compound formulated by our research group, which has been clinically administered for over a decade and has demonstrated the ability to improve the inflammatory state of various acute or chronic kidney diseases. However, the underlying mechanism by which A&P ameliorates renal fibrosis remains unclear.MethodsWe established a mouse model by surgically ligating the unilateral ureter to induce renal injury in vivo. And we utilized renal in situ electroporation of a plasmid with low LncRNA A33 expression to establish the unilateral ureteral obstruction(UUO)mouse model. In vitro, we stimulated primary tubular epithelial cells(pTEC) injury using TGF-β1, siRNA-A33, and pcDNA3.1-A33 plasmids were transfected into pTECs to respectively knockdown and overexpress LncRNA A33, and both in vitro and in vivo models were intervened with A&P.ResultsThe results demonstrated that A&P effectively alleviated renal fibrosis in mice. Subsequent findings indicated high expression of LncRNA A33 in the kidneys of UUO mice and TGF-β1-induced renal tubular cells. In situ, renal electroporation of a plasmid with reduced LncRNA A33 expression revealed that inhibiting LncRNA A33 significantly improved renal fibrosis in UUO mice. Moreover, A&P effectively suppressed LncRNA A33 expression both in vitro and in vivo. Subsequent downregulation of LncRNA A33 in renal tubular epithelial cells resulted in the downregulation of numerous fibrotic markers, a significant inhibition of LncRNA A33, and a notable reduction in downstream ferroptosis signaling. Cell experiments demonstrated that A&P improved renal fibrosis in UUO mice by inhibiting LncRNA A33 and downregulating ferroptosis signaling.ConclusionThrough the inhibition of LncRNA A33 and subsequent downregulation of ferroptosis signaling, A&P showed potential as a therapeutic approach for improving renal fibrosis in UUO mice, providing a potential treatment avenue for CKD.

The role of metabolic memory in diabetic kidney disease: identification of key genes and therapeutic targets.

Diabetic kidney disease (DKD) is characterized by complex pathogenesis and poor prognosis; therefore, an exploration of novel etiological factors may be beneficial. Despite glycemic control, the persistence of transient hyperglycemia still induces vascular complications due to metabolic memory. However, its contribution to DKD remains unclear. Using single-cell RNA sequencing data from the Gene Expression Omnibus (GEO) database, we clustered 12 cell types and employed enrichment analysis and a cell‒cell communication network. Fibrosis, a characteristic of DKD, was found to be associated with metabolic memory. To further identify genes related to metabolic memory and fibrosis in DKD, we combined the above datasets from humans with a rat renal fibrosis model and mouse models of metabolic memory. After overlapping, NDRG1, NR4A1, KCNC4 and ZFP36 were selected. Pharmacology analysis and molecular docking revealed that pioglitazone and resveratrol were possible agents affecting these hub genes. Based on the ex vivo results, NDRG1 was selected for further study. Knockdown of NDRG1 reduced TGF-β expression in human kidney-2 cells (HK-2 cells). Compared to that in patients who had diabetes for more than 10years but not DKD, NDRG1 expression in blood samples was upregulated in DKD patients. In summary, NDRG1 is a key gene involved in regulating fibrosis in DKD from a metabolic memory perspective. Bioinformatics analysis combined with experimental validation provided reliable evidence for identifying metabolic memory in DKD patients.

Costunolide Inhibits Chronic Kidney Disease Development by Attenuating IKKβ/NF-κB Pathway.

Chronic kidney disease (CKD) is a significant worldwide health concern that leads to high mortality rates. The bioactive substance costunolide (CTD) has demonstrated several pharmacological effects and holds promise as a CKD treatment. This study aims to investigate the impact of CTD on CKD and delve into its mechanisms of action. Unilateral ureteral obstruction (UUO) methods and renal fibrosis mice models were created. Various concentrations of CTD were injected into UUO mice models to investigate the therapeutic effects of CTD on renal fibrosis of mice. Then, renal morphology, pathological changes, and the expression of genes related to fibrosis, inflammation and ferroptosis were analysed. RNA sequencing was utilized to identify the main biological processes and pathways involved in renal injury. Finally, both overexpression and inhibition of IKKβ were studied to examine their respective effects on fibrosis and inflammation in both in vitro and in vivo models. CTD treatment was found to significantly alleviate fibrosis, inflammation and ferroptosis in UUO-induced renal fibrosis mice models. The results of RNA sequencing suggested that the IKKβ acted as key regulatory factor in renal injury and the expression of IKKβ was increased in vitro and in vivo renal fibrosis model. Functionally, down-regulated IKKβ expression inhibits ferroptosis, inflammatory cytokine production and collagen deposition. Conversely, IKKβ overexpression exacerbates progressive renal fibrosis. Mechanistically, CTD alleviated renal fibrosis and inflammation by inhibiting the expression of IKKβ and attenuating IKKβ/NF-κB pathway. This study demonstrates that CTD could mitigate renal fibrosis, ferroptosis and inflammation in CKD by modulating the IKKβ/NF-κB pathway, which indicates targeting IKKβ has an enormous potential for treating CKD.

#1110 The AHR antagonist CH223191 alleviates kidney injury and renal fibrosis in adenine-induced chronic kidney disease

Abstract Background and Aims The aryl hydrocarbon receptor (AhR) is a transcription factor that can be activated by a variety of endogenous and exogenous ligands, and is involved in varieties of biological processes. It has been confirmed that the expression of AhR in kidney is elevated in chronic kidney disease (CKD). However, the specific role of AhR signaling pathway in CKD and its molecular mechanism remain unclear. This study aims to investigate the role and underlying mechanism of AhR in CKD, providing potential targets for the treatment of CKD. Method Male C57/B6L mice were randomly divided into control group, model group and treatment group. The model group was fed 0.2% adenine diet for 14 days to establish CKD model, while the treatment group was treated with AhR antagonist CH223191. AhR agonist kynurenine and antagonist CH223191 was applied in mouse tubular epithelial (TCMK-1) cells to investigate the underlying mechanisms of AhR in vitro. The renal function, histological changes and renal fibrosis in mice were evaluated to explore the role of AhR in adenine-induced CKD. Histological changes were evaluated by PAS and MASSON stainings, immunohistochemistry and immunofluorescence. Western blot and qPCR were used to measure protein/gene transcription levels. Results The levels of serum creatinine (p < 0.0001) and urea nitrogen (p < 0.0001) in the model group were significantly increased, with obvious renal tubule dilation and renal fibrosis by PAS and MASSON staining. CH223191 treatment significantly reduced serum creatinine (p=0.0012) and urea nitrogen ( < 0.0001) levels in mice, and alleviated renal fibrosis histologically. Q-PCR and Western-Blot results showed that the renal expression level of AhR in the treatment group was decreased, while the expression levels of fibronectin (Fn) (p < 0.0001), α-smooth muscle actin (α-SMA) (p < 0.0001), type I collagen (p=0.0007) and transforming growth factor-β (TGF-b) (p=0.0027) were significantly decreased. In addition, the expression levels of N-GAL (p < 0.0001) and KIM-1 ( < 0.0001) molecular markers associated with kidney injury were reduced, suggesting that inhibiting AhR expression in vivo can reduce adenine-mediated kidney injury and renal fibrosis in CKD. In vitro experiments indicated that kynurenic acid could induce the expression of AhR and AhR downstream regulatory gene CYP1b1 and upregulated fibrosis-related genes in tubular epithelial cells, while CH223191 treatment downregulated the fibrosis-related genes in tubular epithelial cells. Conclusion AhR mediates renal injury and renal fibrosis in adenine-induced CKD mice. AhR antagonist CH223191 exerts a renal protective effect by reducing the expression of AhR in renal tubular epithelial cells, thereby alleviating renal injury and renal fibrosis.

Activation of BKα channel provented uremic cardiomyopathy by attenuating oxidative stress

Background: Uremic cardiomyopathy and renal fibrosis contribute to chronic kidney disease (CKD)-induced morbidity and mortality. Our previous study found that activation of the large conductance calcium-activated potassium channels (BK channels) attenuated renal fibrosis in mice (Wang et al, Kid Int, 2021). We hypothesized that upregulation of BK channels would suppress cardiac fibrosis in CKD. Methods: CKD mice were induced by 5/6 nephrectomy. To upregulation of BKa channels, BMS-191011 (10 mg/kg BW) was given by IP injection every day for 6-8 weeks. Single channel recordings were used to analyze the activation of BKa channel. Blood pressure was measured by non-invasive photoplethysmography using the tail-cuff method. Echocardiogram was used to assess cardiac function. Cultured H9C2 cardiac myoblasts were used to detect the impact of BK opener (NS1916 20mM) on fibrosis markers and oxidative stress. The mRNA expression of BKa in heart was assayed by qPCR. The Hydrogen peroxide (H2O2) was tested by Amplex Red Hydrogen Peroxide Kit. DHE (Dihydroethidium) was assayed to detect superoxide. Superoxide Dismutase (SOD), a defense of oxidative enzyme, was measured using colorimetric activity kit. Results: The expression of BKa mRNA and protein were decreased and cardiac fibrosis was increased in the heart of CKD mice. Single channel recordings showed that human uremic serum (User) attenuated the activation of BKa channel. Upregulation of BKa channel by BSM-191011 decreased the CKD-induced increase in systolic and diastolic blood pressure. Echocardiogram showed that LV end-diastolic dimension increased in 5/6Nx mice, but back to normal after BK opener treatment. Ejection fraction also improved by this treatment. Fibronectin and vimentin were also significantly increased in CKD heart, whereas BMS treatment attenuated the amount of both proteins. In cultured cardiac myoblasts, User and TGFβ increased fibronectin and collagen I, which were in a dose-dependent manner. BKa openers (NS1916) limited the User and TGF-induced upregulation of both proteins in H9C2 cells. User also significantly increased H2O2 and superoxide production in cultured H9C2 cells. Activation of BKa significantly abolished this upregulation. In addition, User and TGFβ decrease SOD production, NS1916 blocked this decline in a dose dependent manner. Conclusions: Activation of BK channel in CKD animals attenuates cardiac fibrosis likely through reduction of uremia-induced oxidative stress. This study could provide new approaches for developing therapeutic strategies for treating uremic cardiomyopathy in chronic kidney diseases. the Department of Veteran Affairs MERIT Award 5I01BX000994 to HC. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Circadian light/dark cycle reversal exacerbates the progression of chronic kidney disease in mice.

Circadian disruption such as shift work, jet lag, has gradually become a global health issue and is closely associated with various metabolic disorders. The influence and mechanism of circadian disruption on renal injury in chronic kidney disease (CKD) remains inadequately understood. Here, we evaluated the impact of environmental light disruption on the progression of chronic renal injury in CKD mice. By using two abnormal light exposure models to induce circadian disruption, we found that circadian disruption induced by weekly light/dark cycle reversal (LDDL) significantly exacerbated renal dysfunction, accelerated renal injury, and promoted renal fibrosis in mice with 5/6 nephrectomy and unilateral ureteral obstruction (UUO). Mechanistically, RNA-seq analysis revealed significant immune and metabolic disorder in the LDDL-conditioned CKD kidneys. Consistently, renal content of ATP was decreased and ROS production was increased in the kidney tissues of the LDDL-challenged CKD mice. Untargeted metabolomics revealed a significant buildup of lipids in the kidney affected by LDDL. Notably, the level of β-NMN, a crucial intermediate in the NAD+ pathway, was found to be particularly reduced. Moreover, we demonstrated that both β-NMN and melatonin administration could significantly rescue the light-disruption associated kidney dysfunction. In conclusion, environmental circadian disruption may exacerbate chronic kidney injury by facilitating inflammatory responses and disturbing metabolic homeostasis. β-NMN and melatonin treatments may hold potential as promising approaches for preventing and treating light-disruption associated CKD.

A Therapeutic Dose of Lithium Triggers Anti-inflammatory Mechanisms in The Kidneys of Treated Mice

Lithium (Li+) is an effective mood stabilizer in people with schizoaffective disorders with more than 2 million prescriptions per year. Accumulation of Li+ in blood can cause neprotoxicity, manifesting in nephrogenic diabetes insipidus, renal tubular acidosis and tubulointerstitial nephropathy. Damage to renal cells often leads to a local immune response, playing a pivotal role in the ensuing renal injury and subsequent recovery. While both pro- and anti-inflammatory actions of Li+ were reported in various tissues, our understanding of the immunomodulatory effects of Li+ in the kidney is fragmentary. Here, we studied how immune mechanisms contribute to the development of Li+-induced adverse effects in the kidneys of C57BL/6 mice receiving a 0.3% lithium carbonate diet for 28 days. Given the insidious development of Li+-induced renal injury, we hypothesize that nephrotoxic effects of Li+ are alleviated by its anti-inflammatory action. We used fluorescence-activated cell sorting, qPCR, immunoblotting and proteome profiler arrays to assess the pyroptotic inflammatory cascade, characterize the infiltrating immune cells and cytokine markers in the renal tissue isolated from Li+-treated and control mice. We found that both biomarkers of tubular damage, kidney injury marker, KIM-1, and neutrophil gelatinase-associated lipocalin, NGAL, are elevated in the renal tissue of Li+-treated mice. This correlated with a moderate but significant increase in renal fibrosis in mice on Li+ diet. Expression of most genes associated with pyroptotic inflammatory cascade, including NLRP3, Casp-1, IL-1b, was not different in the renal tissue isolated from control and Li+-treated mice. Higher mRNA levels of apoptosis-associated speck-like protein (ASC), in the renal tissue of Li+-treated mice could be attributed to its a pro-apoptotic, rather than inflammasome forming, function. This is in line with elevated levels of CD93 marker in the kidneys of mice on Li+ diet, reflecting increased clearance of apoptotic cells. In general, Li+-treated mice had markedly higher levels of anti-inflammatory cytokine markers in the renal tissue, most notably, IL-10 and adiponectin, and lower abundance of pro-inflammatory markers, such as CD26, CLCX16, PCSK9 and osteopontin. Higher abundance of proinflammatory IL-6 in the kidneys of Li+-treated mice made a notable exception. However, this cytokine can also be released by tubular epithelial cells under insult and induce polarization of anti-inflammatory M2 macrophages. Mice on Li+-diet had higher levels of macrophages (F4.80+ cells) in the renal tissue. Future studies will determine their polarization phenotype. Overall, our results suggest that Li+-induced toxic insult to the tubular epithelial cells is paralleled by the anti-inflammatory immune response localized to the renal tissue. NIDDK 1R01DK125464. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The c-Abl-RACK1-FAK signaling axis promotes renal fibrosis in mice through regulating fibroblast-myofibroblast transition

BackgroundRenal fibrosis is a prevalent manifestation of chronic kidney disease (CKD), and effective treatments for this disease are currently lacking. Myofibroblasts, which originate from interstitial fibroblasts, aggregate in the renal interstitium, leading to significant accumulation of extracellular matrix and impairment of renal function. The nonreceptor tyrosine kinase c-Abl (encoded by the Abl1 gene) has been implicated in the development of renal fibrosis. However, the precise role of c-Abl in this process and its involvement in fibroblast-myofibroblast transition (FMT) remain poorly understood.MethodsTo investigate the effect of c-Abl in FMT during renal fibrosis, we investigated the expression of c-Abl in fibrotic renal tissues of patients with CKD and mouse models. We studied the phenotypic changes in fibroblast or myofibroblast-specific c-Abl conditional knockout mice. We explored the potential targets of c-Abl in NRK-49F fibroblasts.ResultsIn this study, fibrotic mouse and cell models demonstrated that c-Abl deficiency in fibroblasts mitigated fibrosis by suppressing fibroblast activation, fibroblast-myofibroblast transition, and extracellular matrix deposition. Mechanistically, c-Abl maintains the stability of the RACK1 protein, which serves as a scaffold for proteins such as c-Abl and focal adhesion kinase at focal adhesions, driving fibroblast activation and differentiation during renal fibrosis. Moreover, specifically targeting c-Abl deletion in renal myofibroblasts could prove beneficial in established kidney fibrosis by reducing RACK1 expression and diminishing the extent of fibrosis.ConclusionsOur findings suggest that c-Abl plays a pathogenic role in interstitial fibrosis through the regulation of RACK1 protein stabilization and myofibroblast differentiation, suggesting a promising strategy for the treatment of CKD.

Early growth response protein 2 promotes partial epithelial-mesenchymal transition by phosphorylating Smad3 during renal fibrosis

Chronic kidney disease (CKD) is a serious health problem worldwide, which ultimately leads to end-stage renal disease (ESRD). Renal fibrosis is the common pathway and major pathological manifestation for various CKD proceeding to ESRD. However, the underlying mechanisms and effective therapies are still ambiguous. Early growth response 2 (EGR2) is reportedly involved in organ formation and cell differentiation. To determine the role of EGR2 in renal fibrosis, we respectively confirmed the increased expression of EGR2 in kidney specimens from both CKD patients and mice with location in proximal tubules. Genetic deletion of EGR2 attenuated obstructive nephropathy while EGR2 overexpression further promoted renal fibrosis in mice subjected to unilateral ureteral obstruction (UUO) due to extracellular matrix (ECM) deposition mediating by partial epithelial-mesenchymal transition (EMT) as well as imbalance between matrix metalloproteinases (MMPs) and tissue inhibitor of MMPs (TIMPs). We found that EGR2 played a critical role in Smad3 phosphorylation, and inhibition of EGR2 reduced partial EMT leading to blockade of ECM accumulation in cultured human kidney 2 cells (HK2) treated with transforming growth factor β1 (TGF-β1). In addition, the transcription co-stimulator signal transducer and activator of transcription 3 (STAT3) phosphorylation was confirmed to regulate the transcription level of EGR2 in TGF-β1-induced HK2 cells. In conclusion, this study demonstrated that EGR2 played a pathogenic role in renal fibrosis by a p-STAT3-EGR2-p-Smad3 axis. Thus, targeting EGR2 could be a promising strategy for CKD treatment.

Ginsenoside Rg1 attenuates chronic inflammation-induced renal fibrosis in mice by inhibiting AIM2 inflammasome in an Nrf2-dependent manner

Chronic kidney disease (CKD) is a growing global public health issue. Ginsenoside Rg1 (Rg1) has favorable nephroprotective effects, but its role and mechanism in CKD need further investigation. Therefore, we observed the effect of Rg1 on the mouse kidneys after 21 days of low-dose lipopolysaccharide (LPS) exposure. Meanwhile, we investigated the protective mechanism of Rg1 in LPS-induced mesangial cells using the Nrf2 inhibitor ML385. We found that chronic exposure to LPS caused fibrosis in mouse kidneys, accompanied by AIM2 activation and oxidative stress imbalance; however, Rg1 restored the anomalies, accompanied by activation of Nrf2/HO-1 signaling. Finally, our in vitro studies revealed that Rg1′s nephroprotective effects could be inhibited by ML385, and Rg1 can bind Nrf2, suggesting that the protective effects of Rg1 may involve Nrf2 activation and then inhibition of the AIM2. Our study has significant implications for the prevention and treatment of kidney diseases, particularly those related to inflammation.