Mouse Model Of Unilateral Ureteral Obstruction Research Articles

An integrated proteomic and phosphoproteomic landscape of chronic kidney disease.

The prevalence of chronic kidney disease (CKD) is gradually rising worldwide. Patients often remain asymptomatic for an extended period, leaving them unaware of their condition, which can lead to progressing to end-stage renal disease and cause significant economic burden. Improved understanding of CKD pathogenesis can enhance early detection and facilitate advances in drug development. Here, we performed proteomic and phosphoproteomic analyses of the mouse unilateral ureteral obstruction model to explore the molecular mechanisms of chronic kidney injury. 474 significantly differentially expressed proteins and 96 significantly differentially expressed phosphoproteins were screened, respectively. Chronic kidney injury involves complex metabolic pathways such as citrate cycle and hematopoietic system in proteome, and mitochondrial oxidative phosphorylation suppression is a notable alteration. The phosphoproteomic analysis revealed a significant upregulation in epithelial mesenchymal transition and P53 pathways, with a corresponding increase in the phosphorylation of Jun at serine 73. Utilizing HK2 cells, we observed that the reduction oxidative phosphorylation was consistently associated with an augmentation in oxidative stress, which subsequently activated Jun and induced apoptosis. Proteins that act as hubs in these pathways may be candidate targets for CKD intervention. These findings contribute significantly to the current understanding of CKD and provide valuable insights for future studies. SIGNIFICANCE: Chronic kidney disease (CKD) incidence rising annually with varied etiologies, kidney often irreversibly fibrotic, the treatment options are limited and often ineffective due to deficient understanding of renal fibrosis mechanisms. Despite the extensive efforts and numerous omics studies conducted on renal fibrosis, to date, no study has been undertaken to investigate the role of phosphorylated proteins in UUO models. Previously, we performed a comprehensive transcriptome and proteome analysis based on the CKD model, but the potential alterations in the phosphoproteome were not addressed. Here, an integrated proteomic and phosphoproteomic landscape of CKD was completed, which was the the first phosphoproteomic profiles of UUO model. Phosphoproteomic profile suggests that the epithelial mesenchymal transition and P53 pathways is significantly activated in mouse models of kidney injury, and the core protein Jun played a key role in CKD. And a preliminary correlation between P-Jun and oxidative phosphorylation was found base on HK2 cells. Our work contributes to a deeper understanding of the disease characteristics and molecular mechanisms of CKD. Identifying potential CKD targets from proteome and phosphoproteome may provide valuable insights for early diagnosis and treatment of CKD.

Tanshinone I improves renal fibrosis by promoting gluconeogenesis through upregulation of peroxisome proliferator-activated receptor-γ coactivator 1α

Background Renal fibrosis, a hallmark of chronic kidney disease, is closely associated with dysregulated gluconeogenesis. Tanshinone I (Tan I), a bioactive compound derived from the traditional Chinese medicine Danshen, exhibits antifibrotic and anti-inflammatory properties. However, its effects on gluconeogenesis and the mechanisms through which it alleviates renal fibrosis remain unclear. This study aimed to investigate whether Tan I promotes gluconeogenesis and mitigates renal fibrosis. Methods Both in vivo and in vitro experiments were conducted. A unilateral ureteral obstruction (UUO) mouse model was used. Masson’s trichrome, HE, and immunofluorescence staining, along with Western blotting, were employed. Lactate concentrations and a pyruvate tolerance test were conducted to assess glucose metabolism. In vitro, HK2 cells and primary renal tubular cells were treated with transforming growth factor-β (TGFβ) to induce fibrosis, and the effects of Tan I on glucose and lactate levels were examined. Results In the UUO model, Tan I reduced fibrosis, decreased lactate accumulation, and modulated fibrosis markers while upregulating gluconeogenesis markers. Tanshinone I restored impaired renal gluconeogenesis, as evidenced by increased pyruvate levels. In vitro, Tan I inhibited fibrosis, reduced lactate levels, and increased glucose levels in cell supernatants. It also restored gluconeogenesis protein expression and decreased fibrotic protein levels. Peroxisome proliferator-activated receptor-γ coactivator (PGC1α) expression was downregulated in UUO and TGFβ-stimulated models, and Tan I reversed this downregulation. Inhibition of PGC1α in TGFβ-stimulated cells counteracted the antifibrotic and gluconeogenesis-promoting effects of Tan I. Conclusions Tanshinone I ameliorated renal fibrosis by enhancing gluconeogenesis through upregulation of PGC1α.

Enhancement of renal fibrosis in PHF20 transgenic mice.

Plant homeodomain finger protein 20 (PHF20) plays a crucial role in various biological processes, but its involvement in renal fibrosis remains unclear. This study investigated the role of PHF20 in renal fibrosis using a unilateral ureteral obstruction (UUO) mouse model, a widely accepted model for chronic kidney disease. PHF20 transgenic (PHF20-TG) and wild-type (WT) mice were utilized to explore how PHF20 influences renal inflammation and fibrosis. After UUO surgery, serum analysis revealed elevated creatinine levels and increased inflammatory markers, indicating worsened renal function in PHF20-TG mice. Histological analyses, including H&E, PAS, and Sirius Red staining, confirmed significant tissue damage and fibrosis in the PHF20-TG group. Molecular investigations demonstrated enhanced activation of the TGF-β/SMAD2/3 and NF-κB signaling pathways, both of which are crucial in the progression of renal fibrosis. Our findings suggest that PHF20 overexpression accelerates early-stage renal fibrosis by amplifying inflammatory responses and promoting collagen deposition. This indicates that PHF20 expression could serve as an early marker for renal fibrosis progression.

Oral administration of silicon-based agents attenuates renal fibrosis

Renal fibrosis occurs during renal failure progression and is exacerbated by oxidative stress. Oral silicon-based agents generate high intestinal hydrogen levels and reduce systemic oxidative stress. Therefore, in this study, we aimed to verify the efficacy of silicon-based agents against renal fibrosis and elucidate their underlying mechanisms in a unilateral ureteral obstruction mouse model. The mice were grouped based on the treatment provided. Comparisons between the groups were performed on postoperative days 1 and 14. On postoperative day 14, renal fibrosis, tubular cell apoptosis, and transforming growth factor-β and 4-hydroxy-2-nonenal expression were significantly reduced in the silicon-treated group (P < 0.05). Following RNA-sequencing on tissues extracted on postoperative day 14, pathway enrichment analysis was performed, revealing significant downregulation of various pathways associated with tissue fibrosis in mice in the silicon-treated group. Using tissues extracted on postoperative day 1, RNA sequencing and immunohistochemical analysis were performed. We found that complement component 3 expression was significantly reduced in mice treated with a silicon-based agent. Oxidative stress, tubular cell death, pro-fibrotic factors, and renal fibrosis were suppressed in our model following treatment with a silicon-based agent. Moreover, complement component 3 activity was inhibited during the early postoperative phase. Here, we showed that silicon-based agents represent potential novel therapeutic options against renal fibrosis.

Contribution of ECT2 to Tubulointerstitial Fibrosis in the Progression of Chronic Kidney Disease.

Chronic kidney disease (CKD) is a complex disorder resulting from a combination of various environmental and genetic factors. Considerable efforts have been dedicated to elucidating its etiological mechanisms. Nevertheless, the pathogenic mechanism of CKD remains poorly understood, which hinders the development of effective therapeutic strategies. In this study, we aimed to identify novel mediators that could contribute to the development of CKD. The ClinVar, STRING, MEME Suite, TRRUST, bedtools, GEO, and R Studio databases and software were used to analyze their common features and investigate potential CKD disease genes. Transcriptomic analysis, immunohistochemistry, qRT-PCR, and Western blotting were utilized to further validate the role of ECT2 in kidney fibrosis. In total, 26 CKD disease genes were obtained from the ClinVar database, and the STRING, MEME Suite, TRRUST, bedtools, and GEO databases and software were used to analyze their common properties and explore potential CKD disease genes. Epithelial cell transforming sequence 2 (ECT2), cyclin B 1, caspase 7 and collagen alpha-1 (IV) were identified as potential candidates for CKD progression. Weighted correlation network analysis (WGCNA) subsequently revealed the relationships between potential genes and CKD. The results of the transcriptomic analysis further confirmed that ECT2 expression was greater in the kidney tissue of CKD patients than in that of healthy controls. Next, immunohistochemistry and Western blotting demonstrated that ECT2 was predominantly expressed in the renal tubules of a unilateral ureteral obstruction (UUO) mouse model. Consistently, in vitro experiments revealed that ECT2 was upregulated in TGF-β1-treated HK-2 cells. Moreover, ECT2 overexpression or knockdown in HK-2 cells altered the intensity of fibrosis markers. ECT2 significantly affects the development and progression of CKD, particularly in association with tubulointerstitial fibrosis.

β-Mangostin Alleviates Renal Tubulointerstitial Fibrosis via the TGF-β1/JNK Signaling Pathway.

The epithelial-to-mesenchymal transition (EMT) plays a key role in the pathogenesis of kidney fibrosis, and kidney fibrosis is associated with an adverse renal prognosis. Beta-mangostin (β-Mag) is a xanthone derivative obtained from mangosteens that is involved in the generation of antifibrotic and anti-oxidation effects. The purpose of this study was to examine the effects of β-Mag on renal tubulointerstitial fibrosis both in vivo and in vitro and the corresponding mechanisms involved. As shown through an in vivo study conducted on a unilateral ureteral obstruction mouse model, oral β-Mag administration, in a dose-dependent manner, caused a lesser degree of tubulointerstitial damage, diminished collagen I fiber deposition, and the depressed expression of fibrotic markers (collagen I, α-SMA) and EMT markers (N-cadherin, Vimentin, Snail, and Slug) in the UUO kidney tissues. The in vitro part of this research revealed that β-Mag, when co-treated with transforming growth factor-β1 (TGF-β1), decreased cell motility and downregulated the EMT (in relation to Vimentin, Snail, and N-cadherin) and phosphoryl-JNK1/2/Smad2/Smad3 expression. Furthermore, β-Mag co-treated with SB (Smad2/3 kinase inhibitor) or SP600125 (JNK kinase inhibitor) significantly inhibited the TGF-β1-associated downstream phosphorylation and activation of JNK1/2-mediated Smad2 targeting the Snail/Vimentin axis. To conclude, β-Mag protects against EMT and kidney fibrotic processes by mediating the TGF-β1/JNK/Smad2 targeting Snail-mediated Vimentin expression and may have therapeutic implications for renal tubulointerstitial fibrosis.

Pharmacological modulation of transglutaminase 2 in the unilateral ureteral obstruction mouse model

BackgroundTransglutaminase 2 (TG2) is a multifunctional enzyme involved in fibrosis by promoting transforming-growth-factor-β1 and crosslinking of extracellular matrix proteins. These functions are dependent on the open conformation, while the closed state of TG2 can induce vasodilation. We explored the putative protective role of TG2 in its closed state on development of renal fibrosis and blood pressure (BP) regulation. MethodsWe studied the unilateral ureteral obstruction (UUO) mouse model treated with LDN27219, which promotes the closed conformation of TG2. Mice were subjected to 7 days UUO or sham operation and treated with vehicle (n = 10), LDN27219 (15 mg/kg/12 h, n = 9) or candesartan (5 mg/kg/day, n = 10) as a clinically comparator. Renal expression of TG2 and pro-fibrotic mediators were evaluated by Western blotting, qPCR and histology, and BP by tail-cuff measurements. ResultsObstructed kidneys showed increased mRNA and protein expression of fibronectin, collagen 3α1 (Col3α1), α-smooth muscle actin and collagen staining. Despite increased renal TG2 mRNA, protein expression was reduced in all UUO groups, but with increased transamidase activity in the vehicle and candesartan groups. LDN27219 reduced mRNA expression of fibronectin and Col3α1, but their protein expression remained unchanged. In contrast to LDN27219, candesartan lowered BP without affecting expression of pro-fibrotic biomarkers. ConclusionRenal TG2 mRNA and protein expression levels seem dissociated, with transamidase activity being increased. LDN27219 influences kidney pro-fibrotic markers at the mRNA level and attenuates transamidase activity but without affecting collagen content or BP. Our findings suggest that TG2 in its closed conformation has anti-fibrotic effects at the molecular level.

Imperatorin Ameliorates Ferroptosis-Associated Cell Death, Inflammation, and Kidney Fibrosis in a Unilateral Ureteral Obstruction Mouse Model

Imperatorin Ameliorates Ferroptosis-Associated Cell Death, Inflammation, and Kidney Fibrosis in a Unilateral Ureteral Obstruction Mouse Model

Dose-Dependent Nephroprotective Effects of an ALK5 Inhibitor in the Unilateral Ureteral Obstruction (UUO) Mouse Model of Kidney Fibrosis

Dose-Dependent Nephroprotective Effects of an ALK5 Inhibitor in the Unilateral Ureteral Obstruction (UUO) Mouse Model of Kidney Fibrosis

Exosomal let-7b-5p deriving from parietal epithelial cells attenuate renal fibrosis through suppression of TGFβR1 and ARID3a in obstructive kidney disease.

As renal progenitor cells, parietal epithelial cells (PECs) have demonstrated multilineage differentiation potential in response to kidney injury. However, the function of exosomes derived from PECs has not been extensively explored. Immunofluorescent staining of Claudin-1 was used to identify primary PECs isolated from mouse glomeruli. Transmission electron microscopy, nanoparticle tracking analysis, and western blotting were used to characterize the properties of PECs-derived exosomes (PEC-Exo). The therapeutic role of PEC-Exo in tubulointerstitial fibrosis was investigated in the unilateral ureteral obstruction (UUO) mouse model and TGF-β1-stimulated HK-2 cells. High-throughput miRNA sequencing was employed to profile PEC-Exo miRNAs. One of the most enriched miRNAs in PEC-Exo was knocked down by transfecting miRNA inhibitor, and then we investigated whether this candidate miRNA was involved in PEC-Exo-mediated tubular repair. The primary PECs expressed Claudin-1, PEC-Exo was homing to obstructed kidney, and TGF-β1 induced HK-2 cells. PEC-Exo significantly alleviated renal inflammation and ameliorated tubular fibrosis both invivo and invitro. Mechanistically, let-7b-5p, highly enriched in PEC-Exo, downregulated the protein levels of transforming growth factor beta receptor 1(TGFβR1) and AT-Rich Interaction Domain 3A(ARID3a) in tubular epithelial cells (TECs), leading to the inhibition of p21 and p27 to restoring cell cycle. Furthermore, administration of let-7b-5p agomir mitigated renal fibrosis invivo. Our findings demonstrated that PEC-derived exosomes significantly repressed the expression of TGFβR1 and ARID3a by delivering let-7b-5p, thereby alleviating renal fibrosis. This study provides novel insights into the role of PEC-Exo in the repair of kidney injury and new ideas for renal fibrosis intervention.

The JNK-associated leucine zipper protein exerts a protective effect on renal parenchymal injury by limiting the inflammatory secretome in tubular cells

JNK-associated leucine zipper protein (JLP) is a newly identified renal endogenous anti-fibrotic factor that is selectively enriched in renal tubular epithelial cells (TECs). The loss of JLP by TECs is a landmark event that heralds the progression of kidney fibrosis. JLP deficiency ensues a series of pathogenetic cellular processes that are conducive to fibrotic injury. Inflammatory injury is functionally relevant in fibrotic kidneys, and TECs play an essential role in fueling inflammation through aberrant secretions. It is speculated that the loss of JLP in TECs is associated with the relentless inflammation during the development of kidney fibrosis. This study examined the alteration of a panel of inflammatory signatures in TECs under kidney fibrotic circumstances using a Jlp gene-modified unilateral ureteral obstruction (UUO) mouse model or cultured HK-2 cells. It was found that a deficiency of JLP in TECs led to a significant increase in the secretion of inflammatory cytokines including interleukin-1β (IL-1β), tumor necrosis factor (TNF-α), and monocyte chemotactic protein-1 (MCP-1), overactivation of the nuclear factor (NF)-κB/c-Jun N-terminal kinase (JNK) pathway, as well as nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome-mediated pyroptosis in response to pro-fibrotic damage. Additionally, the absence of JLP resulted in enhanced macrophage migration and fibroblast activation as paracrine effects elicited by injured TECs. In conclusion, the loss of JLP in TECs catalyses inflammatory injuries in the development of kidney fibrosis.

Imperatorin ameliorates ferroptotic cell death, inflammation, and renal fibrosis in a unilateral ureteral obstruction mouse model

BackgroundImperatorin is a naturally occurring furocoumarin derivative found in traditional Chinese medicine Angelica dahurica for its anticancer, antihypertensive, and antidiabetic properties. Chronic kidney disease (CKD) is a global health issue, characterized by a high prevalence, significant morbidity and mortality, and a range of related complications. ObjectiveThis study aims to investigate the protective effects of imperatorin treatment and the specific underlying mechanisms in progressive CKD. MethodsImperatorin was orally administrated for 14 consecutive days to mice with unilateral ureteral obstruction (UUO) to investigate the renal pathological alternations, pro-inflammatory mediators, antioxidant response, and ferroptotic death signaling. Imperatorin was also tested in the erastin-induced injury of renal proximal tubular cells (NRK-52E). Cell viability, ferroptosis protein markers, erastin-induced oxidative stress, and lipid peroxidation were assessed. ResultsIn vivo, imperatorin treatment alleviated kidney histology alternations and attenuated the protein expression of fibrotic markers. Furthermore, imperatorin administration reduced inflammatory cell infiltration, and alleviated the oxidative stress burden by downregulating protein markers such as catalase, superoxide dismutase 2 (SOD-2), NADPH oxidase 4 (NOX-4), and thioredoxin reductase 1 (Trxr-1). It also mitigated ferroptosis markers such as glutathione peroxidase 4 (GPX4), solute carrier family 7 member 11/cystine transporter (SLC7A11/xCT), and transferrin receptor 1 (TFR-1), and attenuated renal cell apoptosis. In vitro, imperatorin treatment effectively decreased erastin-induced feroptotic cell death, restored the antioxidant enzyme levels, and mitigated lipid peroxidation as well as the expression of ferroptosis-related markers (XCT, GPX4, and p-p53) in a dose-dependent manner. ConclusionOur finding demonstrated for the first time, that imperatorin treatment holds therapeutic potential in a UUO mouse model of CKD and inhibits the erastin-induced oxidative stress, ferroptosis, and subsequent lipid peroxidation in vitro. This highlights the potential of imperatorin as a future therapeutic target for ferroptosis to improve the progression of CKD.

APP-CD74 axis mediates endothelial cell-macrophage communication to promote kidney injury and fibrosis.

Kidney injuries often carry a grim prognosis, marked by fibrosis development, renal function loss, and macrophage involvement. Despite extensive research on macrophage polarization and its effects on other cells, like fibroblasts, limited attention has been paid to the influence of non-immune cells on macrophages. This study aims to address this gap by shedding light on the intricate dynamics and diversity of macrophages during renal injury and repair. During the initial research phase, the complexity of intercellular communication in the context of kidney injury was revealed using a publicly available single-cell RNA sequencing library of the unilateral ureteral obstruction (UUO) model. Subsequently, we confirmed our findings using an independent dataset from a renal ischemia-reperfusion injury (IRI) model. We treated two different types of endothelial cells with TGF-β and co-cultured their supernatants with macrophages, establishing an endothelial cell and macrophage co-culture system. We also established a UUO and an IRI mouse model. Western blot analysis, flow cytometry, immunohistochemistry and immunofluorescence staining were used to validate our results at multiple levels. Our analysis revealed significant changes in the heterogeneity of macrophage subsets during both injury processes. Amyloid β precursor protein (APP)-CD74 axis mediated endothelial-macrophage intercellular communication plays a dominant role. In the in vitro co-culture system, TGF-β triggers endothelial APP expression, which subsequently enhances CD74 expression in macrophages. Flow cytometry corroborated these findings. Additionally, APP and CD74 expression were significantly increased in the UUO and IRI mouse models. Immunofluorescence techniques demonstrated the co-localization of F4/80 and CD74 in vivo. Our study unravels a compelling molecular mechanism, elucidating how endothelium-mediated regulation shapes macrophage function during renal repair. The identified APP-CD74 signaling axis emerges as a promising target for optimizing renal recovery post-injury and preventing the progression of chronic kidney disease.

Nuclear receptor 4A1 ameliorates renal fibrosis by inhibiting vascular endothelial growth factor A induced angiogenesis in UUO rats

IntroductionAngiogenesis is closely related to renal fibrosis; however, its basic mechanism remains unclear. In our study, we found that nuclear receptor 4A1 (NR4A1) inhibits vascular endothelial growth factor A (VEGFA)-induced angiogenesis, ameliorating renal fibrosis. MethodsWe prepared a renal fibrosis animal model with unilateral ureteral obstruction (UUO) and NR4A1 knockdown UUO mice model, Using Human umbilical vein endothelial cells (HUVECs) to conduct all in vitro experiments. We then detected and analyzed the expression levels of NR4A1 and other genes related to angiogenesis and fibrosis. ResultsThe angiogenesis related genes, such as VEGFA, vascular endothelial growth factor receptor-2 (VEGFR-2), endoglin (CD105), as well as the expression of fibrosis related genes that included, α-smooth muscle actin (α-SMA), Vimentin, and Collagen I are all significantly increased in the UUO rat model. In addition, the expression of NR4A1 of the kidney tissue of UUO rats was significantly reduced. Therefore, according to the above results, we speculated that angiogenesis may exacerbate renal fibrosis and NR4A1 may repress renal fibrosis by inhibiting angiogenesis. To further verify the above results, we used VEGFA to stimulate HUVECs with (or without) overexpression or knockdown of NR4A1. The results showed that with prolonged stimulation using VEGFA, the expression of NR4A1 decreases. Overexpression of NR4A1 significantly inhibits the expression of related indicators of angiogenesis and renal fibrosis. Furthermore, knockdown of NR4A1 induces endothelial cell proliferation and migration; therefore, exacerbating angiogenesis and fibrosis. Finally, the results of NR4A1 knockdown UUO mice showed that knockdown of NR4A1 can aggravating kidney damage and induce the expression of angiogenesis and renal fibrosis related indicators, while UUO can significantly induce kidney damage, angiogenesis and renal fibrosis. When knockdown of NR4A1, renal kidney damage, angiogenesis and fibrosis becomes more severe than UUO. Thus, all of these results indicate that NR4A1 can ameliorate renal fibrosis by inhibiting angiogenesis. ConclusionsNR4A1 can inhibit angiogenesis to ameliorate renal fibrosis.

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.

MiR-92a-3p Knockdown Attenuates Transforming Growth Factor-β1-induced Tubulointerstitial Fibrosis by Targeting LIN28A-mediated EMT Pathway.

The role of microRNAs in regulating tubulointerstitial fibrosis, a key feature of progressive chronic kidney disease, is of significant importance. LIN28A has been reported to attenuate renal fibrosis in obstructive nephropathy. Here, our objective was to investigate the precise biological function of the miR-92a-3p/LIN28A axis in tubulointerstitial fibrosis. The human renal proximal tubular epithelial (HK-2) cell line was exposed to transforming growth factor (TGF)-β1, establishing an in vitro model mimicking tubulointerstitial fibrosis. Luciferase reporter assay was utilized to investigate the relationship between miR-92a-3p and LIN28A. Cell transfection techniques were employed to modify the expression of miR-92a-3p and LIN28A. An in vivo model of tubulointerstitial fibrosis was created by inducing unilateral ureteral obstruction (UUO) in C57BL/6N mice. Our initial observations showed that TGF-β1 treatment of HK-2 cells and the UUO mice model led to an increase in miR-92a-3p expression and a decrease in LIN28A expression. We confirmed that miR-92a-3p directly targeted LIN28A in HK-2 cells. In TGF-β1-stimulated HK-2 cells, knocking down miR-92a-3p notably reduced the levels of alpha smooth muscle actin and vimentin and concurrently enhanced the expression of E-cadherin. These changes were counteracted upon transfection with si-LIN28A. Thus, directing interventions toward miR-92a-3p holds the potential to emerge as a viable therapeutic approach for addressing tubulointerstitial fibrosis.

Modified Fangji Huangqi Decoction alleviates renal interstitial fibrosis by inhibiting TGF-β1/Smad/Snail signaling pathway during epithelial-mesenchymal transition

This study investigated the effects of modified Fangji Huangqi Decoction on the expression of proteins related to epithelial-mesenchymal transition(EMT) in a mouse model of unilateral ureteral obstruction( UUO) and in a rat renal tubular epithelial cell(NRK-52E) model of fibrosis induced by transforming growth factor β1(TGF-β1). It aims to decipher the molecular mechanism by which modified Fangji Huangqi Decoction alleviates renal interstitial fibrosis. C57/BL mice were subjected to UUO.After the surgery, the mice were treated with 0. 5-fold and 2-fold concentrations of modified Fangji Huangqi Decoction and fosinopril sodium(positive control) for 7 days. The interstitial collagen deposition in the kidney was assessed by Masson staining. Western blot and RT-qPCR were employed to determine the expression levels of TGF-β1, phosphorylated Smad2/3(p-Smad2/3), Smad2/3, Snail,epithelial cadherin(E-cadherin), alpha smooth muscle actin(α-SMA), and vimentin. The NRK-52E cell model induced by TGF-β1was treated with the serum samples collected from SD rats treated with different concentrations of modified Fangji Huangqi Decoction.The CCK-8 assay was employed to examine the effects of the serum samples on NRK-52E cell proliferation. The cell morphology in different groups was observed under a microscope. Furthermore, the modeled cells were treated with the serum containing 1-fold decoction. Western blot and RT-qPCR were then employed to measure the expression levels of p-Smad2/3, Smad2/3, Snail,E-cadherin, α-SMA, and vimentin in the cells. Under the same conditions, sh RNA was used to silence the Snail gene, and measurements were repeated before and after treatment with the serum containing 1-fold decoction. The results indicated that modified Fangji Huangqi Decoction alleviated the fibrotic injury in the mouse model of UUO and the fibrosis in the NRK-52E cell model. The treatment with the decoction down-regulated the protein and m RNA levels of EMT-related indicators including p-Smad2/3, α-SMA,Snail, and vimentin, while it up-regulated the expression of E-cadherin. After sh RNA silencing of the Snail gene, the protein and m RNA levels of E-cadherin, α-SMA, and vimentin showed no significant differences before and after treatment with the serum containing the decoction. The results suggest that modified Fangji Huangqi Decoction may alleviate renal interstitial fibrosis by inhibiting the TGF-β1/Smad/Snail signaling pathway and regulating the EMT process.

Wogonin ameliorates ER stress-associated inflammatory response, apoptotic death and renal fibrosis in a unilateral ureteral obstruction mouse model

Wogonin, a vital bioactive compound extracted from the medicinal plant, Scutellaria baicalensis, has been wildly used for its potential in mitigating the progression of chronic diseases. Chronic kidney disease (CKD) represents a significant global health challenge due to its high prevalence, morbidity and mortality rates, and associated complications. This study aimed to assess the potential of wogonin in attenuating renal fibrosis and to elucidate the underlying molecular mechanisms using a unilateral ureteral obstruction (UUO) mouse model as a CKD mimic. Male mice, 8 weeks old, underwent orally administrated of either 50 mg/kg/day of wogonin or positive control of 5 mg/kg/day candesartan following UUO surgery. NRK52E cells were exposed to tumor growth factors−beta (TGF−β) to evaluate the anti-fibrotic effects of wogonin. The results demonstrated that wogonin treatment effectively attenuated TGF-β-induced fibrosis markers in NRK-52E cells. Additionally, administration of wogonin significantly improved histopathological alterations and downregulated the expression of pro-fibrotic factors (Fibronectin, α-smooth muscle actin, Collagen IV, E-cadherin, and TGF-β), oxidative stress markers (Catalase, superoxide dismutase 2, NADPH oxidase 4, and thioredoxin reductase 1), inflammatory molecules (Cyclooxygenase-2 and TNF-α), and the infiltration of neutrophils and macrophages in UUO mice. Furthermore, wogonin treatment mitigated endoplasmic reticulum (ER) stress-associated molecular markers (GRP78, GRP94, ATF4, CHOP, and the caspase cascade) and suppressed apoptosis. The findings indicate that wogonin treatment ameliorates key fibrotic aspects of CKD by attenuating ER stress-related apoptosis, inflammation, and oxidative stress, suggesting its potential as a future therapeutic target.

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.

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.