Unilateral Ureteral Obstruction Mice Research Articles

Immune checkpoint activity exacerbate renal interstitial fibrosis progression by enhancing PD-L1 expression in renal tubular epithelial cells

Renal interstitial fibrosis (RIF) is often associated with inflammatory cell infiltration and no effective therapy. Programmed death cell-1 (PD-1) and its ligand PD-L1 were playing critical roles in T cell coinhibition and exhaustion, but the role in RIF is unclear. Here the data analyses of serum from 122 IgA nephrology (IgAN) patients showed that high level of soluble PD-1(sPD-1) was an independent risk factor for RIF and renal function progression. PD-L1 was also overexpressed in renal interstitial tissues from both IgAN patients with high level of sPD-1 and the unilateral ureteral obstruction (UUO) mouse. PD-L1 was significantly overexpressed in HK-2 cells with upregulated collagen and α-SMA when stimulated by inflammation or hypoxia in vitro. Additionally, matrix metalloproteinases (MMP-2) could increase the level of sPD-1 in culture supernatant when added in co-culture system of HK-2 and jurkat cells, which implied serum sPD-1 of IgAN might be cleaved by MMP-2 from T cells infiltrated into the tubulointerstitial inflammatory microenvironment. Crucially, injection of PD-L1 fusion protein, the blocker of sPD-1, could ameliorate kidney fibrosis in UUO mice by increasing T cell coinhibition and exhaustion, suggesting the therapeutic potential of PD-L1 fusion targeting for renal fibrosis. Take together, it reveals a novel causal role of sPD-1 in serum and PD-L1 of renal interstitial tissues in the development of renal fibrosis of IgAN, and targeting sPD-1 in serum by PD-L1 fusion protein is a potential therapeutic approach to prevent renal fibrosis of IgAN.

Iguratimod prevents renal fibrosis in unilateral ureteral obstruction model mice by suppressing M2 macrophage infiltration and macrophage–myofibroblast transition

Iguratimod is a novel synthetic, small-molecule immunosuppressive agent used to treat rheumatoid arthritis. Through ongoing exploration of its role and mechanisms of action, iguratimod has been observed to have antifibrotic effects in the lung and skin; however, its effect on renal fibrosis remains unknown. This study aimed to investigate whether iguratimod could affect renal fibrosis progression. Three different concentrations of iguratimod (30 mg/kg/day, 10 mg/kg/day, and 3 mg/kg/day) were used to intervene in unilateral ureteral obstruction (UUO) model mice. Iguratimod at 10 mg/kg/day was observed to be effective in slowing UUO-mediated renal fibrosis. In addition, stimulating bone marrow-derived macrophages with IL-4 and/or iguratimod, or with TGF-β and iguratimod or SRC inhibitors in vitro, suggested that iguratimod mitigates the progression of renal fibrosis in UUO mice, at least in part, by inhibiting the IL-4/STAT6 signaling pathway to attenuate renal M2 macrophage infiltration, as well as by impeding SRC activation to reduce macrophage–myofibroblast transition. These findings reveal the potential of iguratimod as a treatment for renal disease.

Exosomes From Human Umbilical Cord Stem Cells Suppress Macrophage-to-myofibroblast Transition, Alleviating Renal Fibrosis.

Renal fibrosis, a progressive scarring of the kidney, lacks effective treatment. Human umbilical cord mesenchymal stem cell-derived exosomes (HucMSC-Exos) hold promise for treating kidney diseases due to their anti-inflammatory properties. This study investigates their potential to lessen renal fibrosis by targeting macrophage-to-myofibroblast transformation (MMT), a key driver of fibrosis.We employed a mouse model of unilateral ureteral obstruction (UUO) and cultured cells exposed to transforming growth factor-β (TGF-β) to mimic MMT.HucMSC-Exos were administered to UUO mice, and their effects on kidney function and fibrosis were assessed. Additionally, RNA sequencing and cellular analysis were performed to elucidate the mechanisms by which HucMSC-Exos inhibit MMT. HucMSC-Exos treatment significantly reduced kidney damage and fibrosis in UUO mice. They downregulated markers of fibrosis (Collagen I, vimentin, alpha-smooth muscle actin) and suppressed MMT (α-SMA + F4/80 + cells). Furthermore, ARNTL, a specific molecule, emerged as a potential target of HucMSC-Exos in hindering MMT and consequently preventing fibrosis.HucMSC-Exos effectively lessen renal fibrosis by suppressing MMT, suggesting a novel therapeutic strategy for managing kidney damage and fibrosis.

Antisense oligonucleotide-mediated terminal intron retention of endoglin: A potential strategy to inhibit renal interstitial fibrosis

TGF-β is considered an important cytokine in the development of interstitial fibrosis in chronic kidney disease. The TGF-β co-receptor endoglin (ENG) tends to be upregulated in kidney fibrosis. ENG has two membrane bound isoforms generated via alternative splicing. Long-ENG was shown to enhance the extent of renal fibrosis in an unilateral ureteral obstruction mouse model, while short-ENG inhibited renal fibrosis. Here we aimed to achieve terminal intron retention of endoglin using antisense-oligo nucleotides (ASOs), thereby shifting the ratio towards short-ENG to inhibit the TGF-β1-mediated pro-fibrotic response. We isolated mRNA from kidney biopsies of patients with chronic allograft disease (CAD) (n = 12) and measured total ENG and short-ENG mRNA levels. ENG mRNA was upregulated 2.3 fold (p < 0.05) in kidneys of CAD patients compared to controls, while the percentage short-ENG of the total ENG mRNA was significantly lower (1.8 fold; p < 0.05). Transfection of ASOs that target splicing regulatory sites of ENG into TK173 fibroblasts led to higher levels of short-ENG (2 fold; p < 0.05). In addition, we stimulated these cells with TGF-β1 and measured a decrease in upregulation of ACTA2, COL1A1 and FN1 mRNA levels, and protein expression of αSMA, collagen type I, and fibronectin. These results show a potential for ENG ASOs as a therapy to reduce interstitial fibrosis in CKD.

Icariin alleviates renal inflammation and tubulointerstitial fibrosis via Nrf2-mediated attenuation of mitochondrial damage.

Tubulointerstitial fibrosis is an inevitable consequence of all progressive chronic kidney disease (CKD) and contributes to a substantial health burden worldwide. Icariin, an active flavonoid glycoside obtained from Epimedium species, exerts potential antifibrotic effect. The study aimed to explore the protective effects of icariin against tubulointerstitial fibrosis in unilateral ureteral obstruction (UUO)-induced CKD mice and TGF-β1-treated HK-2 cells, and furthermore, to elucidate the underlying mechanisms. The results demonstrated that icariin significantly improved renal function, alleviated tubular injuries, and reduced fibrotic lesions in UUO mice. Furthermore, icariin suppressed renal inflammation, reduced oxidative stress as evidenced by elevated superoxide dismutase activity and decreased malondialdehyde level. Additionally, TOMM20 immunofluorescence staining and transmission electron microscope revealed that mitochondrial mass and morphology of tubular epithelial cells in UUO mice was restored by icariin. In HK-2 cells treated with TGF-β1, icariin markedly decreased profibrotic proteins expression, inhibited inflammatory factors, and protected mitochondria along with preserving mitochondrial morphology, reducing reactive oxygen species (ROS) and mitochondrial ROS (mtROS) overproduction, and preserving membrane potential. Further investigations demonstrated that icariin could activate nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) pathway both in vivo and in vitro, whereas inhibition of Nrf2 by ML385 counteracted the protective effects of icariin on TGF-β1-induced HK-2 cells. In conclusion, icariin protects against renal inflammation and tubulointerstitial fibrosis at least partly through Nrf2-mediated attenuation of mitochondrial dysfunction, which suggests that icariin could be developed as a promising therapeutic candidate for the treatment of CKD.

RfxCas13d-mediated inhibition of Circ1647 alleviates renal fibrosis via PI3K/AKT signaling pathway

Background Circular RNAs (CircRNAs) have been shown to be involved in the development of chronic kidney disease (CKD). This study aimed to investigate the role of Circ1647 in renal fibrosis, which is a hallmark of CKD. Methods In this study, we established a unilateral ureteral obstruction (UUO) model and delivered Circ1647 RfxCas13d knockdown plasmid into renal parenchymal cells via retrograde injection through the ureter followed by electroporation. After that, the pathological changes were determined by Hematoxylin and Eosin. Meanwhile, Immunohistochemistry, qRT-PCR and Western blot were conducted to assess the degree of fibrosis. In addition, overexpressing of Circ1647 in renal tubular epithelial cells (TCMK1) was performed to investigate the underlying mechanisms of Circ1647. Results Our results displayed that electroporation-mediated knockdown of Circ1647 by RfxCas13d knockdown plasmid significantly inhibited renal fibrosis in UUO mice as evidenced by reduced expression of fibronectin and α-SMA (alpha-smooth muscle actin). Conversely, overexpression of Circ1647 in TCMK1 cells promoted the fibrosis. In terms of mechanism, Circ1647 may mediate the PI3K/AKT Signaling Pathway as demonstrated by the balance of the phosphorylation of PI3K and AKT in vivo and the aggravated phosphorylation of PI3K and AKT in vitro. These observations were corroborated by the effects of the PI3K inhibitor LY294002, which mitigated fibrosis post Circ1647 overexpression. Conclusion Our study suggests that Circ1647 plays a significant role in renal fibrosis by mediating the PI3K/AKT signaling pathway. RfxCas13d-mediated inhibition of Circ1647 may serve as a therapeutic target for renal fibrosis in CKD.

Abstract 2969: Targeting novel senescence markers by conditionally active biologics eliminates senescence-associated secretory phenotype in in vitro and in vivo models

Abstract Clearing senescent cells has been shown to have promising effects against cancer and age-related pathologies in preclinical models as well as early human clinical trials. However, current senescent cell elimination strategies are focused on targets that do not distinguish between hyper-inflamed or SASP senescent cells versus relatively non-inflamed senescent cells. The use of senolytics in the clinic is still limited due to their cytotoxicity to either normal cells or potentially beneficial senescent cells. A more effective senescence cell targeted approach is needed to reduce side effects and increase the efficiency of senescence cell removal or reduction. Conditionally Active Biologics (CAB) technology is a proprietary platform unique in its ability to be selectively active in the context of diseased tissues, but not normal tissues. The aberrant accumulation of senescent cells in aged and cancerous tissue triggers inflammatory signaling through a senescence-associated secretory phenotype (SASP), promoting aging and tumor progression. Since our CAB technology is currently being tested as a cancer therapy by targeting cell surface markers such as AXL, ROR2, and CTLA4 in clinical studies, we explored whether CAB technology allows selective removal of senescent cells in SASP-associated microenvironments. In our previous report, we demonstrated several novel senescence-specific surface antigens upregulated in senescent cells (1). CAB antibodies targeting these senescence markers have no or very low binding to the target antigen on senescence cells in physiological, alkaline conditions, but have strong binding in glycolytic, acidic SASP conditions in in vitro binding assays. In in vitro antibody-dependent cell-mediated cytotoxicity assays, CAB antibodies were more potent against senescent cells compared to proliferating cells and displayed high selectivity for the glycolytic, acidic condition. To study whether CAB antibody reduces senescent cell occurrence in vivo, we developed a unilateral ureteral obstruction (UUO) mouse model to induce senescent cells, fibrosis, and inflammation in mouse kidney. The mice treated with CAB antibodies had a significant reduction of senescent cells and inflammatory cells infiltrated in the renal cortex, compared to the mice treated with benchmark and isotype antibodies in UUO kidneys. Thus, the CAB antibody-based therapeutic is a promising strategy for senescent cell removal in an inflammatory microenvironment. In conclusion, CAB technology provides a new generation of biologics with an increased safety margin and therapeutic index targeting SASP, acidic senescence cells in cancer and age-related diseases. (1). Chen J, Wang J, Liu H, Chang C, Boyle WJ, and Short JM. Abstract 4795: Conditionally active biologics eliminates senescence cells in cancer and aging. Cancer Res (2023) 83. Citation Format: Jian Chen, Jian Wang, Haizheng Liu, Cathy Chang, William J. Boyle, Jay M. Short. Targeting novel senescence markers by conditionally active biologics eliminates senescence-associated secretory phenotype in in vitro and in vivo models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2969.

FTO-mediated m6A mRNA demethylation aggravates renal fibrosis by targeting RUNX1 and further enhancing PI3K/AKT pathway.

Chronic kidney disease (CKD) is a global health burden, with ineffective therapies leading to increasing morbidity and mortality. Renal interstitial fibrosis is a common pathway in advanced CKD, resulting in kidney function and structure deterioration. In this study, we investigate the role of FTO-mediated N6-methyladenosine (m6A) and its downstream targets in the pathogenesis of renal fibrosis. M6A modification, a prevalent mRNA internal modification, has been implicated in various organ fibrosis processes. We use a mouse model of unilateral ureteral obstruction (UUO) as an invivo model and treated tubular epithelial cells (TECs) with transforming growth factor (TGF)-β1 as invitro models. Our findings revealed increased FTO expression in UUO mouse model and TGF-β1-treated TECs. By modulating FTO expression through FTO heterozygous mutation mice (FTO+/- ) invivo and small interfering RNA (siRNA) invitro, we observed attenuation of UUO and TGF-β1-induced epithelial-mesenchymal transition (EMT), as evidenced by decreased fibronectin and N-cadherin accumulation and increased E-cadherin levels. Silencing FTO significantly improved UUO and TGF-β1-induced inflammation, apoptosis, and inhibition of autophagy. Further transcriptomic assays identified RUNX1 as a downstream candidate target of FTO. Inhibiting FTO was shown to counteract UUO/TGF-β1-induced RUNX1 elevation invivo and invitro. We demonstrated that FTO signaling contributes to the elevation of RUNX1 by demethylating RUNX1 mRNA and improving its stability. Finally, we revealed that the PI3K/AKT pathway may be activated downstream of the FTO/RUNX1 axis in the pathogenesis of renal fibrosis. In conclusion, identifying small-molecule compounds that target this axis could offer promising therapeutic strategies for treating renal fibrosis.

Microvesicles from Mesenchymal Stem CellsOverexpressing MiR-34a Ameliorate Renal Fibrosis In Vivo.

We recently discovered that microvesicles (MVs) derived from mesenchymal stem cells (MSCs) overexpressing miRNA-34a can alleviate experimental kidney injury in mice. In this study, we further explored the effects of miR34a-MV on renal fibrosis in the unilateral ureteral obstruction (UUO) models. Methods. Bone marrow MSCs were modified by lentiviruses overexpressing miR-34a, and MVs were collected from the supernatants of MSCs. C57BL6/J mice were divided into control, unilateral ureteral obstruction (UUO), UUO + MV, UUO + miR-34aMV and UUO + miR-34a-inhibitor-MV groups. MVs were injected to mice after surgery. The mice were then euthanized on day 7 and 14 of modeling, and renal tissues were collected for further analyses by Hematoxylin and eosin, Masson's trichrome, and Immunohistochemical (IHC) staining. Results. The UUO + MV group exhibited a significantly reduced degree of renal interstitial fibrosis with inflammatory cell infiltration, tubular epithelial cell atrophy, and vacuole degeneration compared with the UUO group. Surprisingly, overexpressing miR-34a enhanced these effects of MSC-MV on the UUO mice. Conclusion. Our study demonstrates that miR34a further enhances the effects of MSC-MV on renal fibrosis in mice through the regulation of epithelial-to-mesenchymal transition (EMT) and Notch pathway. miR-34a may be a candidate molecular therapeutic target for the treatment of renal fibrosis. DOI: 10.52547/ijkd.7673.

Hypermethylation leads to the loss of HOXA5, resulting in JAG1 expression and NOTCH signaling contributing to kidney fibrosis

Epigenetic regulations, including DNA methylation, are critical to the development and progression of kidney fibrosis, but the underlying mechanisms remain elusive. Here, we show that fibrosis of the mouse kidney was associated with the induction of DNA methyltransferases and increases in global DNA methylation and was alleviated by the DNA methyltransferase inhibitor 5-Aza-2’-deoxycytidine (5-Aza). Genome wide analysis demonstrated the hypermethylation of 94 genes in mouse unilateral ureteral obstruction kidneys, which was markedly reduced by 5-Aza. Among these genes, Hoxa5 was hypermethylated at its gene promoter and this hypermethylation was associated with reduced HOXA5 expression in fibrotic mouse kidneys after ureteral obstruction or unilateral ischemia-reperfusion injury. 5-Aza prevented Hoxa5 hypermethylation, restored HOXA5 expression, and suppressed kidney fibrosis. Down-regulation of HOXA5 was verified in human kidney biopsies from patients with chronic kidney disease and correlated with the increased kidney fibrosis and DNA methylation. Kidney fibrosis was aggravated by conditional knockout of Hoxa5 and alleviated by conditional knockin of Hoxa5 in kidney proximal tubules of mice. Mechanistically, we found that HOXA5 repressed Jag1 transcription by directly binding to its gene promoter, resulting in the suppression of JAG1-NOTCH signaling during kidney fibrosis. Thus, our results indicate that loss of HOXA5 via DNA methylation contributes to fibrogenesis in kidney diseases by inducing JAG1 and consequent activation of the NOTCH signaling pathway.

C/EBPα aggravates renal fibrosis in CKD through the NOX4-ROS-apoptosis pathway in tubular epithelial cells

BackgroundChronic kidney disease (CKD) is a prevalent renal disorder with various risk factors. Emerging evidence indicates that the transcriptional factor CCAAT/enhancer binding protein alpha (C/EBPα) may be associated with renal fibrosis. However, the precise role of C/EBPα in CKD progression remains unexplored. MethodsWe investigated the involvement of C/EBPα in CKD using two distinct mouse models induced by folic acid (FA) and unilateral ureteral obstruction (UUO). Additionally, we used RNA sequencing and KEGG analysis to identify potential downstream pathways governed by C/EBPα. FindingsCebpa knockout significantly shielded mice from renal fibrosis and reduced reactive oxygen species (ROS) levels in both the FA and UUO models. Primary tubular epithelial cells (PTECs) lacking Cebpa exhibited reduced apoptosis and ROS accumulation following treatment with TGF-β. RNA sequencing analysis suggested that apoptosis is among the primary pathways regulated by C/EBPα, and identified NADPH oxidoreductase 4 (NOX4) as a key protein upregulated upon C/EBPα induction (ICCB280). Treatment with l-Theanine, a potential NOX4 inhibitor, mitigated renal fibrosis and inflammation in both the FA and UUO mouse models. InterpretationOur study unveils a role for C/EBPα in suppressing renal fibrosis, mitigating ROS accumulation, and reducing cell apoptosis. Furthermore, we investigate whether these protective effects are mediated by C/EBPα's regulation of NOX4 expression. These findings present a promising therapeutic target for modulating ROS and apoptosis in renal tubular cells, potentially offering an approach to treating CKD and other fibrotic diseases.

Noninvasive Assessment of Kidney Injury by Combining Structure and Function Using Artificial Intelligence-Based Manganese-Enhanced Magnetic Resonance Imaging.

Contrast-enhanced magnetic resonance imaging (MRI) is seriously limited in kidney injury detection due to the nephrotoxicity of clinically used gadolinium-based contrast agents. Herein, we propose a noninvasive method for the assessment of kidney injury by combining structure and function information based on manganese (Mn)-enhanced MRI for the first time. As a proof of concept, the Mn-melanin nanoprobe with good biocompatibility and excellent T1 relaxivity is applied in MRI of a unilateral ureteral obstruction mice model. The abundant renal structure and function information is obtained through qualitative and quantitative analysis of MR images, and a brand new comprehensive assessment framework is proposed to precisely identify the degree of kidney injury successfully. Our study demonstrates that Mn-enhanced MRI is a promising approach for the highly sensitive and biosafe assessment of kidney injury in vivo.

Retraction: Interferon gamma peptidomimetic targeted to interstitial myofibroblasts attenuates renal fibrosis after unilateral ureteral obstruction in mice.

Retraction: Interferon gamma peptidomimetic targeted to interstitial myofibroblasts attenuates renal fibrosis after unilateral ureteral obstruction in mice.

Altenusin, a fungal metabolite, alleviates TGF-β1-induced EMT in renal proximal tubular cells and renal fibrosis in unilateral ureteral obstruction

Renal fibrosis is a pathological feature of chronic kidney disease (CKD), progressing toward end-stage kidney disease (ESKD). The aim of this study is to investigate the therapeutic potential of altenusin, a farnesoid X receptor (FXR) agonist derived from fungi, on renal fibrosis. The effect of altenusin was determined (i) in vitro using the transforming growth factor β1 (TGF-β1)-induced epithelial to mesenchymal transition (EMT) of human renal proximal tubular cells and (ii) in vivo using mouse unilateral ureteral obstruction (UUO). The findings revealed that incubation of 10 ng/ml TGF-β1 promotes morphological change in RPTEC/TERT1 cells, a human renal proximal tubular cell line, from epithelial to fibroblast-like cells. TGF-β1 markedly increased EMT markers namely α-smooth muscle actin (α-SMA), fibronectin, and matrix metalloproteinase 9 (MMP-9), while decreased the epithelial marker E-cadherin. Co-incubation TGF-β1 with altenusin preserved the epithelial characteristics of the renal epithelial cells by antagonizing TGF-β/Smad signaling pathway, specifically a decreased phosphorylation of Smad2/3 with an increased level of Smad7. Interestingly, the antagonizing effect of altenusin does not require FXR activation. Moreover, altenusin could reverse TGF-β1-induced fibroblast-like cells to epithelial-like cells. Treatment on UUO mice with 30 mg/kg altenusin significantly reduced the expression of α-SMA, fibronectin, and collagen type 1A1 (COL1A1). The reduction in the renal fibrosis markers is correlated with the decreased phosphorylation of Smad2/3 levels but does not improve E-cadherin protein expression. Collectively, altenusin reduces EMT in human renal proximal tubular cells and renal fibrosis by antagonizing the TGF-β/Smad signaling pathway.

A Natural Small Molecule Mitigates Kidney Fibrosis by Targeting Cdc42-mediated GSK-3β/β-catenin Signaling.

Kidney fibrosis is a common fate of chronic kidney diseases (CKDs), eventually leading to renal dysfunction. Yet, no effective treatment for this pathological process has been achieved. During the bioassay-guided chemical investigation of the medicinal plant Wikstroemia chamaedaphne, a daphne diterpenoid, daphnepedunin A (DA), is characterized as a promising anti-renal fibrotic lead. DA shows significant anti-kidney fibrosis effects in cultured renal fibroblasts and unilateral ureteral obstructed mice, being more potent than the clinical trial drug pirfenidone. Leveraging the thermal proteome profiling strategy, cell division cycle 42 (Cdc42) is identified as the direct target of DA. Mechanistically, DA targets to reduce Cdc42 activity and down-regulates its downstream phospho-protein kinase Cζ(p-PKCζ)/phospho-glycogen synthase kinase-3β (p-GSK-3β), thereby promoting β-catenin Ser33/37/Thr41 phosphorylation and ubiquitin-dependent proteolysis to block classical pro-fibrotic β-catenin signaling. These findings suggest that Cdc42 is a promising therapeutic target for kidney fibrosis, and highlight DA as a potent Cdc42 inhibitor for combating CKDs.

The E3 ubiquitin ligase TRIM39 modulates renal fibrosis induced by unilateral ureteral obstruction through regulating proteasomal degradation of PRDX3

Renal fibrosis is considered to be the ultimate pathway for various chronic kidney disease, with a complex etiology and great therapeutic challenges. Tripartite motif-containing (TRIM) family proteins have been shown to be involved in fibrotic diseases, but whether TRIM39 plays a role in renal fibrosis remain unexplored. In this study, we investigated the role of TRIM39 in renal fibrosis and its molecular mechanism. TRIM39 expression was analyzed in patients’ specimens, HK-2 cells and unilateral ureteral obstruction (UUO) mice were used for functional and mechanistic studies. We found an upregulated expression of TRIM39 in renal fibrosis human specimens and models. In addition, TRIM39 knockdown was found efficient for alleviating renal fibrosis in both UUO mice and HK-2 cells. Mechanistically, we demonstrated that TRIM39 interacted with PRDX3 directly and induced ubiquitination degradation of PRDX3 at K73 and K149 through the K48 chain, which resulted in ROS accumulation and increased inflammatory cytokine generation, and further aggravated renal fibrosis. It provided an emerging potential target for the therapies of renal fibrosis.

Downregulating ANGPTL3 by miR-144-3p promoted TGF-β1-induced renal interstitial fibrosis via activating PI3K/AKT signaling pathway

Despite observations of decreased ANGPTL3 (angiopoietin-like protein 3) levels in tubular atrophy and renal interstitial fibrosis (RIF), its functional implications and regulatory mechanisms in RIF remain unclear. This investigation employed unilateral ureteral obstruction (UUO) mice as in vivo model and human proximal kidney tubuloepithelial HK-2 cells under TGF-β1 treatment as in vitro model to explore RIF. The RIF extent was evaluated using H & E staining and Masson's trichrome staining. There was a significant decrease in ANGPTL3 levels and an increase in miR-144-3p, accompanied by heightened expressions of α-SMA, p-PI3K, p-AKT, Collagen I, and Fibronectin in the UUO mice and HK-2 cells treated with TGF-β1. Enhancing ANGPTL3 expression or suppressing miR-144-3p mitigated TGF-β1-induced cellular apoptosis, inflammation, and PI3K/AKT signaling pathway activation, as evidenced by altered levels of α-SMA, Collagen I, Fibronectin, and associated signaling markers. Using a bioinformatics approach, a miR-144-3p binding site was discovered on the ANGPTL3 mRNA, and this finding was subsequently confirmed through luciferase reporter assay. In HK-2 cells stimulated with TGF-β1, the suppression of ANGPTL3 negated the effects of inhibiting miR-144-3p. Under comparable conditions, the use of LY294002, an inhibitor of the PI3K/AKT pathway, nullified the effects caused by the knockdown of ANGPTL3. Collectively, these findings indicate that miR-144-3p exacerbates RIF through PI3K/AKT pathway activation by targeting ANGPTL3, highlighting a novel potential therapeutic target for RIF management.

CXC chemokine receptor 7 ameliorates renal fibrosis by inhibiting β-catenin signaling and epithelial-to-mesenchymal transition in tubular epithelial cells

Renal fibrosis is a common feature of various chronic kidney diseases. However, the underlying mechanism remains poorly understood. The CXC chemokine receptor (CXCR) family plays a role in renal fibrosis; however, the detailed mechanisms have not been elucidated. In this study, we investigated the potential role of CXCR7 in mediating renal fibrosis. CXCR7 expression is decreased in unilateral ischemia–reperfusion injury (UIRI) and unilateral ureteral obstruction mouse models. Furthermore, CXCR7 was specifically expressed primarily in the Lotus Tetragonolobus Lectin-expressing segment of tubules, was slightly expressed in the peanut agglutinin-expressing segment, and was barely expressed in the Dolichos biflorus agglutinin-expressing segment. Administration of pFlag-CXCR7, an overexpression plasmid for CXCR7, significantly inhibited the activation of β-catenin signaling and protected against the progression of epithelial-to-mesenchymal transition (EMT) and renal fibrosis in a UIRI mouse model. Using cultured HKC-8 cells, we found that CXCR7 significantly downregulated the expression of active β-catenin and fibrosis-related markers, including fibronectin, Collagen I, and α-SMA. Furthermore, CXCR7 significantly attenuated TGF-β1-induced changes in β-catenin signaling, EMT and fibrosis. These results suggest that CXCR7 plays a crucial role in inhibiting the activation of β-catenin signaling and the progression of EMT and renal fibrosis. Thus, CXCR7 could be a novel therapeutic target for renal fibrosis.

Dual soluble epoxide hydrolase inhibitor - farnesoid X receptor agonist interventional treatment attenuates renal inflammation and fibrosis.

Renal fibrosis associated with inflammation is a critical pathophysiological event in chronic kidney disease (CKD). We have developed DM509 which acts concurrently as a farnesoid X receptor agonist and a soluble epoxide hydrolase inhibitor and investigated DM509 efficacy as an interventional treatment using the unilateral ureteral obstruction (UUO) mouse model. Male mice went through either UUO or sham surgery. Interventional DM509 treatment (10mg/kg/d) was started three days after UUO induction and continued for 7 days. Plasma and kidney tissue were collected at the end of the experimental protocol. UUO mice demonstrated marked renal fibrosis with higher kidney hydroxyproline content and collagen positive area. Interventional DM509 treatment reduced hydroxyproline content by 41% and collagen positive area by 65%. Renal inflammation was evident in UUO mice with elevated MCP-1, CD45-positive immune cell positive infiltration, and profibrotic inflammatory gene expression. DM509 treatment reduced renal inflammation in UUO mice. Renal fibrosis in UUO was associated with epithelial-to-mesenchymal transition (EMT) and DM509 treatment reduced EMT. UUO mice also had tubular epithelial barrier injury with increased renal KIM-1, NGAL expression. DM509 reduced tubular injury markers by 25-50% and maintained tubular epithelial integrity in UUO mice. Vascular inflammation was evident in UUO mice with 9 to 20-fold higher ICAM and VCAM gene expression which was reduced by 40-50% with DM509 treatment. Peritubular vascular density was reduced by 35% in UUO mice and DM509 prevented vascular loss. Interventional treatment with DM509 reduced renal fibrosis and inflammation in UUO mice demonstrating that DM509 is a promising drug that combats renal epithelial and vascular pathological events associated with progression of CKD.

Kidney Injury by Unilateral Ureteral Obstruction in Mice Lacks Sex Differences.

Renal fibrosis is a critical event in the development and progression of chronic kidney disease (CKD), and it is considered the final common pathway for all types of CKD. The prevalence of CKD is higher in females; however, males have a greater prevalence of end-stage renal disease. In addition, low birth weight and low nephron number are associated with increased risk for CKD. This study examined the development and severity of unilateral ureter obstruction (UUO)-induced renal fibrosis in male and female wild-type (ROP +/+) and mutant (ROP Os/+) mice, a mouse model of low nephron number. Male and female ROP +/+ and ROP Os/+ mice were subjected to UUO, and kidney tissue was collected at the end of the 10-day experimental period. Kidney histological analysis and mRNA expression determined renal fibrosis, tubular injury, collagen deposition, extracellular matrix proteins, and immune cell infiltration. Male and female UUO mice demonstrated marked renal injury, kidney fibrosis, and renal extracellular matrix production. Renal fibrosis and α-smooth muscle actin were increased to a similar degree in ROP +/+ and ROP Os/+ mice with UUO of either sex. There were also no sex differences in renal tubular cast formation or renal infiltration of macrophage in ROP +/+ and ROP Os/+ UUO mice. Interestingly, renal fibrosis and α-smooth muscle actin were 1.5-3-fold greater in UUO-ROP +/+ compared to UUO-ROP Os/+ mice. Renal inflammation phenotypes following UUO were also 30-45% greater in ROP +/+ compared to ROP Os/+ mice. Likewise, expression of extracellular matrix and renal fibrotic genes was greater in UUO-ROP +/+ mice compared to UUO-ROP Os/+ mice. In contrast to these findings, ROP Os/+ mice with UUO demonstrated glomerular hypertrophy with 50% greater glomerular tuft area compared to ROP +/+ with UUO. Glomerular hypertrophy was not sex-dependent in any of the genotypes of ROP mice. These findings provide evidence that low nephron number contributes to UUO-induced glomerular hypertrophy in ROP Os/+ mice but does not enhance renal fibrosis, inflammation, and renal tubular injury. Taken together, we demonstrate that low nephron number contributes to enhanced glomerular hypertrophy but not kidney fibrosis and tubular injury. We also demonstrate that none of the changes caused by UUO was affected by sex in any of the ROP mice genotypes.