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Objective: The purpose of this study was to investigate the role and possible mechanism of lncRNA XIST in renal fibrosis and to provide potential endogenous targets for renal fibrosis in obstructive nephropathy (ON)., Methods: The study included 50 cases of ON with renal fibrosis (samples taken from patients undergoing nephrectomy due to ON) and 50 cases of normal renal tissue (samples taken from patients undergoing total or partial nephrectomy due to accidental injury, congenital malformations, and benign tumors). Treatment of human proximal renal tubular epithelium (HK-2) cells with TGF-β1 simulated renal fibrosis in vitro. Cell viability and proliferation were measured by CCK-8 and EdU, and cell migration was measured by transwell. XIST, miR-124-3p, ITGB1, and epithelial-mesenchymal transition (EMT)-related proteins (E-cadherin, α-SMA, and fibronectin) were detected by PCR and immunoblot. The targeting relationship between miR-124-3p and XIST or ITGB1 was verified by starBase and dual luciferase reporter gene experiments. In addition, The left ureter was ligated in mice as a model of unilateral ureteral obstruction (UUO), and the renal histopathology was observed by HE staining and Masson staining., Results: ON patients with renal fibrosis had elevated XIST and ITGB1 levels and reduced miR-124-3p levels. The administration of TGF-β1 exhibited a dose-dependent promotion of HK-2 cell viability, proliferation, migration, and EMT. Conversely, depleting XIST or enhancing miR-124-3p hindered HK-2 cell viability, proliferation, migration, and EMT in TGF-β1-damaged HK-2 cells HK-2 cells. XIST functioned as a miR-124-3p sponge. Additionally, miR-124-3p negatively regulated ITGB1 expression. Elevating ITGB1 weakened the impact of XIST depletion on TGF-β1-damaged HK-2 cells. Down-regulating XIST improved renal fibrosis in UUO mice., Conclusion: XIST promotes renal fibrosis in ON by elevating miR-124-3p and reducing ITGB1 expressions., Competing Interests: Declaration of competing interest None., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Renal fibrosis (RF) is a typical pathological presentation of end-stage chronic kidney disease (CKD) and autosomal dominant polycystic kidney disease (ADPKD). However, the precise regulatory mechanisms governing this re-expression process remain unclear. Differentially expressed microRNAs (miRNAs) associated with RF were screened by microarray analysis using the Gene Expression Omnibus (GEO) database. The miRNAs upstream of the genes in question were predicted using the miRWalk database. The miRNAs involved in the two GEO data sets were intersected to identify key miRNAs; their regulatory pathways were investigated using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. Subsequently, the effects and the underlying mechanisms of target miRNA on RF were examined in a unilateral ureteral obstruction (UUO)-induced mice renal fibrotic model and a transforming growth factor-β1 (TGF-β1)-induced tubular epithelium (HK-2) fibrotic cell model. In total, 109 and 32 differentially expressed miRNAs were identified in the GSE133530 and GSE80247 data sets, respectively. GREM1 was identified as a hub gene, where its 2196 upstream miRNAs were predicted; miR-574-5p was found to be downregulated and closely related to fibrosis after data set intersection and enrichment analyses, thus was selected for further investigation. A differential expression heatmap (GSE162794) showed that miR-542-5p was downregulated. The expression of GREM1 mRNA was upregulated, whereas that of miR-542-5p was downregulated in UUO mice and fibrotic HK-2 cells as compared with the relevant controls. The binding site of miR-542-5p was predicted at the 3'UTR region of GREM1 and was confirmed by subsequent dual luciferase reporter gene assay. Western blot analysis showed that Gremlin-1 and Fibronectin were significantly upregulated after induction of TGF-β1; when miR-542-5p was overexpressed or GREM1 mRNA was interfered, the upregulations of Gremlin-1 and Fibronectin were significantly reduced. Our research demonstrates that miR-542-5p plays a critical role in the progression of RF, and thus may be a promising therapeutic target for CKD and ADPKD., (© 2024 The Author(s). Journal of Biochemical and Molecular Toxicology published by Wiley Periodicals LLC.)

Background/aim: Angiotensinogen (AGT), a precursor of angiotensin II (AngII), contributes to regulating (patho)physiological conditions, including blood pressure changes, inflammation, and kidney fibrosis. However, the precise role of tissue-specific AGT in kidney fibrosis independent of blood pressure remains to be fully understood. This study investigated the source of intrarenal AGT and its role in kidney injury and fibrosis during obstructive nephropathy., Materials and Methods: Proximal tubule- (PT, major source secreting AGT in the kidney; PKO) or liver- (major source of circulating AGT; LKO) AGT knockout (KO) mice were subjected to unilateral ureteral obstruction (UUO), a blood pressure-independent fibrosis model., Results: UUO increased AGT mRNA and protein levels in the kidneys. PKO decreased AGT mRNA, but LKO enhanced it in UUO kidneys compared with the control. In contrast, the intrarenal protein levels of AGT increased in PKO, but not in LKO in UUO kidneys, indicating that the liver is a major source of intrarenal AGT protein. Expression of megalin, a PT receptor involved in the uptake of circulating AGT, was down-regulated in UUO kidneys and was independent of PKO or LKO. However, none of these changes prevented UUO-induced tubular injury and kidney fibrosis., Conclusion: Hepatic and proximal tubule AGT play distinct roles in contributing to intrarenal AGT levels during UUO, and their genetic inhibitions fail to prevent kidney injury and fibrosis, suggesting a highly complicated signaling pathway of the renin-angiotensin system and an associated compensatory mechanism in obstructive nephropathy., (Copyright © 2024, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

RhoA and its effectors, the transcriptional coactivators myocardin-related transcription factor (MRTF) and serum response factor (SRF), control epithelial phenotype and are indispensable for profibrotic epithelial reprogramming during fibrogenesis. Context-dependent control of RhoA and fibrosis-associated changes in its regulators, however, remain incompletely characterized. We previously identified the guanine nucleotide exchange factor GEF-H1 as a central mediator of RhoA activation in renal tubular cells exposed to inflammatory or fibrotic stimuli. Here we found that GEF-H1 expression and phosphorylation were strongly elevated in two animal models of fibrosis. In the Unilateral Ureteral Obstruction mouse kidney fibrosis model, GEF-H1 was upregulated predominantly in the tubular compartment. GEF-H1 was also elevated and phosphorylated in a rat pulmonary artery banding (PAB) model of right ventricular fibrosis. Prolonged stimulation of LLC-PK 1 tubular cells with tumor necrosis factor (TNF)-α or transforming growth factor (TGF)-β1 increased GEF-H1 expression and activated a luciferase-coupled GEF-H1 promoter. Knockdown and overexpression studies revealed that these effects were mediated by RhoA, cytoskeleton remodeling, and MRTF, indicative of a positive feedback cycle. Indeed, silencing endogenous GEF-H1 attenuated activation of the GEF-H1 promoter. Of importance, inhibition of MRTF using CCG-1423 prevented GEF-H1 upregulation in both animal models. MRTF-dependent increase in GEF-H1 was prevented by inhibition of the transcription factor Sp1, and mutating putative Sp1 binding sites in the GEF-H1 promoter eliminated its MRTF-dependent activation. As the GEF-H1/RhoA axis is key for fibrogenesis, this novel MRTF/Sp1-dependent regulation of GEF-H1 abundance represents a potential target for reducing renal and cardiac fibrosis. NEW & NOTEWORTHY We show that expression of the RhoA regulator GEF-H1 is upregulated in tubular cells exposed to fibrogenic cytokines and in animal models of kidney and heart fibrosis. We identify a pathway wherein GEF-H1/RhoA-dependent MRTF activation through its noncanonical partner Sp1 upregulates GEF-H1. Our data reveal the existence of a positive feedback cycle that enhances Rho signaling through control of both GEF-H1 activation and expression. This feedback loop may play an important role in organ fibrosis.

The long noncoding RNA growth arrest-specific 5 (lncRNA Gas5) is implicated in various kidney diseases. In this study, we investigated the lncRNA Gas5 expression profile and its critical role as a potential biomarker in the progression of chronic kidney disease. Subsequently, we assessed the effect of lncRNA Gas5 deletion on renal fibrosis induced by unilateral ureteral obstruction (UUO). The results indicated that loss of lncRNA Gas5 exacerbates UUO-induced renal injury and extracellular matrix deposition. Notably, the deletion of lncRNA Gas5 had a similar effect on control mice. The fibrogenic phenotype observed in mice lacking lncRNA Gas5 correlates with peroxisome proliferator-activated receptor (PPAR) signaling pathway activation and aberrant cytokine and chemokine reprogramming. Single-cell RNA sequencing analysis revealed key transcriptomic features of fibroblasts after Gas5 deletion, revealing heterogeneous cellular states suggestive of a propensity for renal fibrosis. Our findings indicate that lncRNA Gas5 regulates the differentiation and activation of immune cells and the transcription of key genes in the PPAR signaling pathway. These data offer novel insights into the involvement of lncRNA Gas5 in renal fibrosis, potentially paving the way for innovative diagnostic and therapeutic targets., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Rationale: Acute kidney injury (AKI) has substantial rates of mortality and morbidity, coupled with an absence of efficacious treatment options. AKI commonly transits into chronic kidney disease (CKD) and ultimately culminates in end-stage renal failure. The interferon-stimulated gene 15 (ISG15) level was upregulated in the kidneys of mice injured by ischemia-reperfusion injury (IRI), cisplatin, or unilateral ureteral obstruction (UUO), however, its role in AKI development and subsequent AKI-to-CKD transition remains unknown. Methods: Isg15 knockout ( Isg15 KO) mice challenged with bilateral or unilateral IRI, cisplatin, or UUO were used to investigate its role in AKI. We established cellular models with overexpression or knockout of ISG15 and subjected them to hypoxia-reoxygenation, cisplatin, or transforming growth factor- β1 (TGF-β1) stimulation. Renal RNA-seq data obtained from AKI models sourced from public databases and our studies, were utilized to examine the expression profiles of ISG15 and its associated genes. Additionally, published single cell RNA-seq data from human kidney allograft biopsies and mouse IRI model were analyzed to investigate the expression patterns of ISG15 and the type I TGF-β receptor (TGFβR1). Western blotting, qPCR, co-immunoprecipitation, and immunohistochemical staining assays were performed to validate our findings. Results: Alleviated pathological injury and renal function were observed in Isg15 KO mice with IRI-, cisplatin-, or UUO-induced AKI and the following AKI-to-CKD transition. In hypoxia-reoxygenation, cisplatin or TGF-β1 treated HK-2 cells, knockout ISG15 reduced stimulus-induced cell fibrosis, while overexpression of ISG15 with modification capacity exacerbated cell fibrosis. Immunoprecipitation assays demonstrated that ISG15 promoted ISGylation of TGFβR1, and inhibited its ubiquitination. Moreover, knockout of TGFβR1 blocked ISG15's fibrosis-exacerbating effect in HK-2 cells, while overexpression of TGFβR1 abolished the renal protective effect of ISG15 knockout during IRI-induced kidney injury. Conclusions: ISG15 plays an important role in the development of AKI and subsequent AKI-to-CKD transition by promoting TGFβR1 ISGylation., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Progranulin (PGRN) may have two opposing effects-inflammation and anti-inflammation-in different diseases. Although previous studies have reported that PGRN is involved in liver fibrosis, its involvement in tubulointerstitial fibrosis remains to be fully elucidated. Herein, we investigated these issues using PGRN-knockout (KO) mice treated with unilateral ureteral obstruction (UUO). Eight-week-old male PGRN-KO and wild-type (WT) mice were euthanized 3 and 7 days following UUO, and their kidneys were harvested for histopathological analysis. The renal expression of PGRN was evaluated by immunohistochemical and/or western blot analyses. The renal mRNA levels of markers related to inflammation (Il1b, Tnf, Il6, Ccl2, and Adgre1) and fibrosis (Tgfb1, Acta2, Fn1, and Col1a2) were evaluated using quantitative PCR. Histological changes such as renal tubular atrophy, urinary casts, and tubulointerstitial fibrosis were significantly improved in UUO-KO mice compared with UUO-WT mice. Quantitative PCR revealed that the mRNA expression levels of all inflammation- and fibrosis-related markers were lower in UUO-KO mice than in UUO-WT mice at 3 and/or 7 days after UUO. Moreover, PGRN and GRN protein levels were higher in the kidneys of UUO-WT mice than in mice that did not undergo UUO. Elevated GRN levels associated with excess PGRN levels may be involved in the occurrence of renal inflammation and fibrosis in UUO mice.

Abstract: 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., (Copyright © 2024 Copyright: © 2024 Journal of Physiological Investigation.)

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. Downregulation 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., (Copyright © 2024 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

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.

Introduction: Angiogenesis 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., Methods: We 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., Results: The 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., Conclusions: NR4A1 can inhibit angiogenesis to ameliorate renal fibrosis., Competing Interests: Declaration of competing interest The authors have declared that no competing interest exists., (Copyright © 2024 Elsevier B.V. All rights reserved.)

OBJECTIVE：To elucidate the mechanism by which Huoxue Jiedu Huayu recipe (, HJHR) regulates angiogenesis in the contralateral kidney of unilateral ureteral obstruction (UUO) rats and the mechanism by which it reduces of renal fibrosis., Methods: Male Wistar rats were randomly divided into 4 groups: the sham group, UUO group (180 d of left ureter ligation), UUO plus eplerenone (EPL) group, and UUO plus HJHR group. After 180 d of oral drug administration, blood and contralateral kidneys were collected for analysis. Angiogenesis- and fibrosis-related indexes were detected., Results: HJHR and EPL improved structural damage and renal interstitial fibrosis in the contralateral kidney and reduced the protein expression levels of α-smooth muscle actin (α-SMA), vimentin and collagen I. Moreover, these treatments could reduce the expression of vascular endothelial growth factor-A (VEGFA) by inhibiting the infiltration of macrophages. Furthermore, HJHR and EPL significantly reduced the expression of CD34 and CD105 by downregulating VEGFA production, which inhibited angiogenesis. Finally, the coexpressions of CD34, CD105 and α-SMA were decreased in the HJHR and EPL groups, indicating that endothelial-to-mesenchymal transition was inhibited., Conclusions: These findings confirm that HJHR alleviates contralateral renal fibrosis by inhibiting VEGFA-induced angiogenesis, encourage the use of HJHR against renal interstitial fibrosis and provide a theoretical basis for the clinical management of patients with CKD.

Renal fibrosis (RF) stands as a pivotal pathological process in the advanced stages of chronic kidney disease (CKD), and impeding its progression is paramount for delaying the advancement of CKD. The miR-10 family, inclusive of miR-10a and miR-10b, has been implicated in the development of various fibrotic diseases. Nevertheless, the precise role of miR-10 in the development of RF remains enigmatic. In this study, we utilized both an in vivo model involving unilateral ureteral obstruction (UUO) in mice and an in vitro model employing TGF-β1 stimulation in HK-2 cells to unravel the mechanism underlying the involvement of miR-10a/b in RF. The findings revealed heightened expression of miR-10a and miR-10b in the kidneys of UUO mice, accompanied by a substantial increase in p-Smad3 and renal fibrosis-related proteins. Conversely, the deletion of these two genes led to a notable reduction in p-Smad3 levels and the alleviation of RF in mouse kidneys. In the in vitro model of TGF-β1-stimulated HK-2 cells, the co-overexpression of miR-10a and miR-10b fostered the phosphorylation of Smad3 and RF, while the inhibition of miR-10a and miR-10b resulted in a decrease in p-Smad3 levels and RF. Further research revealed that miR-10a and miR-10b, through binding to the 3'UTR region of Vasohibin-1 (VASH-1), suppressed the expression of VASH-1, thereby promoting the elevation of p-Smad3 and exacerbating the progression of RF. The miR-10 family may play a pivotal role in RF.

Effective therapies of chronic kidney disease (CKD) are lacking due to the unclear molecular pathogenesis. Previous single omics-studies have described potential molecular regulation mechanism of CKD only at the level of transcription or translation. Therefore, this study generated an integrated transcriptomic and proteomic profile to provide deep insights into the continuous transcription-translation process during CKD. The comprehensive datasets identified 14,948 transcripts and 6423 proteins, 233 up-regulated and 364 down-regulated common differentially expressed genes of transcriptome and proteome were selected to further combined bioinformatics analysis. The obtained results revealed reactive oxygen species (ROS) metabolism and antioxidant system due to imbalance of mitochondria and peroxisomes were significantly repressed in CKD. Overall, this study presents a valuable multi-omics analysis that sheds light on the molecular mechanisms underlying CKD. SIGNIFICANCE: Chronic kidney disease (CKD) is a progressive and irreversible condition that results in abnormal kidney function and structure, and is ranked 18th among the leading causes of death globally, leading to a significant societal burden. Hence, there is an urgent need for research to detect new, sensitive, and specific biomarkers. Omics-based studies offer great potential to identify underlying disease mechanisms, aid in clinical diagnosis, and develop novel treatment strategies for CKD. Previous studies have mainly focused on the regulation of gene expression or protein synthesis in CKD, thereby compelling us to conduct a meticulous analysis of transcriptomic and proteomic data from the UUO mouse model. Here, we have performed a unified analysis of CKD model by integrating transcriptomes and protein suites for the first time. Our study contributes to a deeper understanding of the pathogenesis of CKD and provides a basis for subsequent disease management and drug development., Competing Interests: Declaration of competing interest All the authors of this paper declare that we have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.There is no professional or other personal interest of any nature or kind in any product, service or company that could be construed as influencing the position presented in, or the review of the manuscript entitled., (Copyright © 2023. Published by Elsevier B.V.)

Background: The hedgehog signaling pathway exerts vital functions in regulating epithelial-to-mesenchymal transition (EMT) in renal interstitial fibrosis (RIF). It was reported that lncRNA-maternally expressed gene 3 (lncRNA Meg3) can regulate hepatic fibrosis by regulating the expression of smoothened (Smo) in the hedgehog signaling pathway. However, the specific role of lncRNA Meg3 in renal fibrosis resulting from unilateral ureteral obstruction (UUO) by regulating the hedgehog signaling pathway has not been reported. Hence, this research aimed to expound the effects of lncRNA Meg3 on renal fibrosis induced by UUO in rats via the hedgehog pathway., Methods: Peripheral blood was collected from patients with chronic kidney disease (CKD, CKD group) and healthy volunteers (Normal group) at the same period. In addition, 6-week-old male Sprague-Dawley (SD) rats were divided to Sham, UUO, UUO+shRNA Negative control (shNC), and UUO+sh-Meg3 groups, and their kidney tissues and serum were gathered. Next, quantitative real-time polymerase chain reaction (qRT-PCR) was employed for detecting the lncRNA Meg3 expression level in the serum of patients and renal tissue of rats; kits for testing levels of blood urea nitrogen (BUN), creatinine (Cr), hydroxyproline (HYP), and 24-hour urine protein (24-up) in rats of each group; hematoxylin and eosin (HE) staining and Masson staining for observing kidney tissue and renal fibrosis level in rats; western blot for measuring levels of collagen type III (Col III), α-Smooth muscle actin (α-SMA), fibronectin, E-cadherin, sonic hedgehog (Shh), patched (Ptch) protein, smoothened (Smo) protein and glioma-associated oncogene homolog 1 (Gli1) protein expression., Results: LncRNA Meg3 was highly expressed in CKD patients and UUO rats ( p < 0.01). In contrast to the UUO+shNC group, knocking down lncRNA Meg3 improved renal injury, relieved pathological renal lesions, and reduced kidney fibrosis and related protein levels. It inhibited the hedgehog pathway in kidney tissues of UUO rats ( p < 0.05 and p < 0.01)., Conclusions: LncRNA Meg3 can aggravate UUO-induced rat renal fibrosis by activating the hedgehog pathway.

The molecular basis of renal interstitial fibrosis, a major pathological feature of progressive kidney diseases, remains poorly understood. Autophagy has been implicated in renal fibrosis, but whether it promotes or inhibits fibrosis remains controversial. Moreover, it is unclear how autophagy is activated and sustained in renal fibrosis. The present study was designed to address these questions using the in vivo mouse model of unilateral ureteral obstruction and the in vitro model of hypoxia in renal tubular cells. Both models showed the activation of hypoxia-inducible factor-1 (HIF-1) and autophagy along with fibrotic changes. Inhibition of autophagy with chloroquine reduced renal fibrosis in unilateral ureteral obstruction model, whereas chloroquine and autophagy-related gene 7 knockdown decreased fibrotic changes in cultured renal proximal tubular cells, supporting a profibrotic role of autophagy. Notably, pharmacological and genetic inhibition of HIF-1 led to the suppression of autophagy and renal fibrosis in these models. Mechanistically, knock down of BCL2 and adenovirus E1B 19-kDa-interacting protein 3 ( BNIP3 ), a downstream target gene of HIF , decreased autophagy and fibrotic changes during hypoxia in BUMPT cells. Together, these results suggest that HIF-1 may activate autophagy via BNIP3 in renal tubular cells to facilitate the development of renal interstitial fibrosis. NEW & NOTEWORTHY Autophagy has been reported to participate in renal fibrosis, but its role and underlying activation mechanism is unclear. In this study, we report the role of HIF-1 in autophagy activation in models of renal fibrosis and further investigate the underlying mechanism.

Introduction: Long noncoding RNA (lncRNA) cancer susceptibility candidate 2 (CASC2) alleviates the progression of diabetic nephropathy by inhibiting inflammation and fibrosis. This study investigated how CASC2 impacts renal interstitial fibrosis (RIF) through regulating M1 macrophage (M1) polarization., Method: Nine-week-old mice underwent unilateral ureteral obstruction (UUO) establishment. Macrophages were induced toward M1 polarization using lipopolysaccharide (LPS) in vitro and cocultured with fibroblasts to examine how M1 polarization influences RIF. LnCeCell predicted that CASC2 interacted with myocyte enhancer factor 2 C (MEF2C), which was validated by dual-luciferase reporter assay. CASC2/MEF2C overexpression was achieved by lentivirus-expressing lncRNA CASC2 injection in vivo or CASC2 and MEF2C transfection in vitro. Renal injury was evaluated through biochemical analysis and hematoxylin-eosin/Masson staining. Macrophage infiltration and M1 polarization in the kidney and/or macrophages were detected by immunofluorescence, flow cytometry, and/or quantitative reverse transcription polymerase chain reaction (qRT-PCR). Expressions of CASC2, MEF2C, and markers related to inflammation/M1/fibrosis in the kidney/macrophages/fibroblasts were analyzed by qRT-PCR, fluorescence in situ hybridization, enzyme-linked immunosorbent assay, and/or Western blot., Result: In the kidneys of mice, CASC2 was downregulated and macrophage infiltration was promoted time-dependently from days 3 to 14 post-UUO induction; CASC2 overexpression alleviated renal histological abnormalities, hindered macrophage infiltration and M1 polarization, downregulated renal function markers serum creatinine and blood urea nitrogen and inflammation/M1/fibrosis-related makers, and offset UUO-induced MEF2C upregulation. LncRNA CASC2 overexpression inhibited fibroblast fibrosis and M1 polarization in cocultured fibroblasts with LPS-activated macrophages. Also, CASC2 bound to MEF2C and inhibited its expression in LPS-activated macrophages. Furthermore, MEF2C reversed the inhibitory effects of lncRNA CASC2 overexpression., Conclusion: CASC2 alleviates RIF by inhibiting M1 polarization through directly downregulating MEF2C expression. CASC2 might represent a promising value of future investigations on treatment for RIF., (© 2023 S. Karger AG, Basel.)

Introduction: Several mouse models with diverse disease etiologies are used in preclinical research for chronic kidney disease (CKD). Here, we performed a head-to-head comparison of renal transcriptome signatures in standard mouse models of CKD to assess shared and distinct molecular changes in three mouse models commonly employed in preclinical CKD research and drug discovery., Methods: All experiments were conducted on male C57BL/6J mice. Mice underwent sham, unilateral ureter obstruction (UUO), or unilateral ischemic-reperfusion injury (uIRI) surgery and were terminated two- and 6-weeks post-surgery, respectively. The adenine-supplemented diet-induced (ADI) model of CKD was established by feeding with adenine diet for 6 weeks and compared to control diet feeding. For all models, endpoints included plasma biochemistry, kidney histology, and RNA sequencing., Results: All models displayed increased macrophage infiltration (F4/80 IHC) and fibrosis (collagen 1a1 IHC). Compared to corresponding controls, all models were characterized by an extensive number of renal differentially expressed genes (≥11,000), with a notable overlap in transcriptomic signatures across models. Gene expression markers of fibrosis, inflammation, and kidney injury supported histological findings. Interestingly, model-specific transcriptome signatures included several genes representing current drug targets for CKD, emphasizing advantages and limitations of the three CKD models in preclinical target and drug discovery., Conclusion: The UUO, uIRI, and ADI mouse models of CKD have significant commonalities in their renal global transcriptome profile. Model-specific renal transcriptional signatures should be considered when selecting the specific model in preclinical target and drug discovery., (© 2024 S. Karger AG, Basel.)

Renal interstitial fibrosis (RIF) represents an irreversible and progressive pathological manifestation of chronic renal disease, which ultimately leads to end-stage renal disease. Long noncoding RNAs (lncRNAs) have been suggested to be involved in the progression of RIF. Small nucleolar RNA host gene 16 (SNHG16), a member of lncRNAs, has been found to be involved in the progression of pulmonary fibrosis. This paper first researched the effect of SNHG16 on renal fibrosis. We established a unilateral ureteral obstruction (UUO)-induced mouse RIF model by ligation of the left ureter to evaluate the biological function of SNHG16 in RIF. As a result, SNHG16 was upregulated in UUO-induced renal fibrotic tissues. Knockdown of SNHG16 inhibited RIF and reduced alpha-smooth muscle actin (α-SMA), fibronectin, and college IV expression. miR-205 was a target of SNHG16, and downregulated in UUO-induced renal fibrotic tissues. Inhibition of miR-205 promoted RIF and increased the expression of α-SMA, college IV, and fibronectin. Overexpression of SNHG16 promoted the UUO-induced RIF, but miR-205 abrogated this effect of SNHG16. Histone deacetylase 5 (HDAC5) showed high expression in UUO-induced renal fibrotic tissues. Knockdown of HDAC5 significantly reduced α-SMA, fibronectin, and college IV expression in renal tissues of UUO-induced mice. Inhibition of miR-205 promoted HDAC5 expression, but knockdown of SNHG16 inhibited HDAC5 expression in renal tissues of UUO-induced mice. In conclusion, SHNG16 is highly expressed in renal fibrotic tissues of UUO-induced mice. Knockdown of SHNG16 may prevent UUO-induced RIF by indirectly upregulating HDAC5 via targeting miR-205. SHNG16 may be novel target for treating renal fibrosis., (© 2023 Wiley Periodicals LLC.)

Background: Renal fibrosis is a basic pathological change of almost all chronic kidney disorders. Epithelial-mesenchymal transition (EMT) and excessive extracellular matrix (ECM) accumulation play a crucial role in the process of fibrosis., Methods: Western blot and qRT-PCR were accomplished to analyze the expression levels of target proteins and genes, respectively. The fibrotic levels in the renal tissues of rats were confirmed utilizing Masson staining. Expression of ECM-related α-SMA in the renal tissues was determined by immunohistochemistry assay. The combination of GRB2 associated binding protein 1 (GAB1) and miR-200a was ensured by starBase database and luciferase reporter assay., Results: Our data uncovered that miR-200a was downregulated, but GAB1 was upregulated in the renal tissues of the rat experienced unilateral ureteral obstruction (UUO). Overexpression of miR-200a improved tissues fibrosis, suppressed GAB1 expression and ECM deposition, and inactivated Wnt/β-catenin in UUO rats. Moreover, miR-200a expression was inhibited, while GAB1 expression was facilitated in the TGF-β1-induced HK-2 cells. In TGF-β1-induced HK-2 cells, miR-200a overexpression inhibited GAB1 expression, also declined ECM-related proteins and mesenchymal markers expression. Oppositely, miR-200a overexpression facilitated epithelial marker expression in the TGF-β1-induced HK-2 cells. Next, the data revealed that miR-200a inhibited GAB1 expression through binding to the mRNA 3'-UTR of GAB1. Increasing of GAB1 reversed the regulation of miR-200a to GAB1 expression, Wnt/β-catenin signaling activation, EMT and ECM accumulation., Conclusion: Overall, miR-200a increasing improved renal fibrosis through attenuating EMT and ECM accumulation by limiting Wnt/β-catenin signaling via sponging GAB1, indicating miR-200a may be a promising objective for renal disease therapy., (Copyright © 2023 Sociedad Española de Nefrología. Published by Elsevier España, S.L.U. All rights reserved.)

Background: Chronic kidney disease (CKD) is a significant global health issue characterized by progressive loss of kidney function. Renal interstitial fibrosis (TIF) is a common feature of CKD, but current treatments are seldom effective in reversing TIF. Nicotinamide N-methyltransferase (NNMT) has been found to increase in kidneys with TIF, but its role in renal fibrosis is unclear., Methods: Using mice with unilateral ureteral obstruction (UUO) and cultured renal interstitial fibroblast cells (NRK-49F) stimulated with transforming growth factor-β1 (TGF-β1), we investigated the function of NNMT in vivo and in vitro ., Results: We performed single-cell transcriptome sequencing (scRNA-seq) on the kidneys of mice and found that NNMT increased mainly in fibroblasts of UUO mice compared to sham mice. Additionally, NNMT was positively correlated with the expression of renal fibrosis-related genes after UUO injury. Knocking down NNMT expression reduced fibroblast activation and was accompanied by an increase in DNA methylation of p53 and a decrease in its phosphorylation., Conclusions: Our findings suggest that chronic kidney injury leads to an accumulation of NNMT, which might decrease p53 methylation, and increase the expression and activity of p53. We propose that NNMT promotes fibroblast activation and renal fibrosis, making NNMT a novel target for preventing and treating renal fibrosis., Competing Interests: All authors declare that they have no competing interests., (© 2023 Zhen et al.)

Renal interstitial fibrosis (RIF) considered the primary irreversible cause of chronic kidney disease. Recently, accumulating studies demonstrated that lncRNAs play an important role in the pathogenesis of RIF. However, the underlying exact mechanism of lncRNA MALAT1 in RIF remains barely known. Here, the aim of our study was to investigate the dysregulate expression of lncRNA MALAT1 in TGF-β1 treated HK2/NRK-49F cells and unilateral ureteral obstruction (UUO) mice model, defining its effects on HK2/NRK-49F cells and UUO mice fibrosis process through the miR-124-3p/ITGB1 signaling axis. It was found that lncRNA MALAT1 and ITGB1 was significantly overexpression, while miR-124-3p was downregulated in HK2/NRK-49F cells induced by TGF-β1 and in UUO mice model. Moreover, knockdown of lncRNA MALAT1 remarkably downregulated the proteins level of fibrosis-related markers, ITGB1, and upregulated the expression of epithelial marker E-cadherin. Consistently, mechanistic studies showed that miR-124-3p can directly binds to lncRNA MALAT1 and ITGB1. And the protect effect of Len-sh-MALAT1 on fibrosis related protein levels could be partially reversed by co-transfected with inhibitor-miR-124-3p. Moreover, the expression trend of LncRNA MALAT1/miR-124-3p/ITGB1 in renal tissues of patients with obstructive nephropathy (ON) was consistent with the results of cell and animal experiments. Taken together, these results indicated that lncRNA MALAT1 could promote RIF process in vitro and in vivo via the miR-124-3p/ITGB1 signaling pathway. These findings suggest a new regulatory pathway involving lncRNA MALAT1, which probably serves as a potential therapeutic target for RIF., (© 2023. Springer Nature Limited.)

Tubulointerstitial fibrosis is considered the final convergent pathway of progressive chronic kidney diseases (CKD) regardless of etiology. However, mechanisms underlying kidney injury-induced fibrosis largely remain unknown. Recent studies have indicated that transcriptional intermediary factor 1γ (TIF1γ) inhibits the progression of fibrosis in other organs. Here, we found that TIF1γ was highly expressed in the cytoplasm and nucleus of the kidney proximal tubule. Interestingly, we found tubular TIF1γ expression was decreased in patients with CKD, including those with diabetes, hypertension, and IgA nephropathy, and in mouse models with experimental kidney fibrosis (unilateral ureteral obstruction [UUO], folic acid nephropathy [FAN], and aristolochic acid-induced nephrotoxicity). Tubule-specific knock out of TIF1γ in mice exacerbated UUO- and FAN-induced tubular cell polyploidy and subsequent fibrosis, whereas overexpression of kidney TIF1γ protected mice against kidney fibrosis. Mechanistically, in tubular epithelial cells, TIF1γ exerted an antifibrotic role via transforming growth factor-β (TGF-β)-dependent and -independent signaling. TIF1γ hindered TGF-β signaling directly by inhibiting the formation and activity of the transcription factor Smad complex in tubular cells, and we discovered that TIF1γ suppressed epidermal growth factor receptor (EGFR) signaling upstream of TGF-β signaling in tubular cells by ubiquitylating EGFR at its lysine 851/905 sites thereby promoting EGFR internalization and lysosomal degradation. Pharmacological inhibition of EGFR signaling attenuated exacerbated polyploidization and the fibrotic phenotype in mice with tubule deletion of TIF1γ. Thus, tubular TIF1γ plays an important role in kidney fibrosis by suppressing profibrotic EGFR and TGF-β signaling. Hence, our findings suggest that maintaining homeostasis of tubular TIF1γ may be a new therapeutic option for treating tubulointerstitial fibrosis and subsequent CKD., (Copyright © 2023 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Background and Objectives: Congenital ureteral stenosis is one of the leading causes of impaired urinary drainage and subsequent dilatation of the urinary collecting system, known as hydronephrosis or ureterohydronephrosis. The mechanism that leads to obstruction is not clearly known. Multiple studies in rat models have shown increased angiotensin II and TGFβ levels in obstructed ureteral tissue. The aim of the study is to investigate the expression of fibrosis-related genes in obstructive and normal ureteral tissue. Material and Methods: It is a monocentric pilot study in which nineteen patients were selected prospectively. 17 patients underwent Hynes-Anderson pyeloplasty due to the PUJO; two patients underwent ureteroneocystostomy due to ureterovesical junction obstruction (UVJO); and six patients were chosen for the control group: five underwent nephrectomies due to the kidney tumor and one underwent upper pole heminephrectomy due to the duplex kidney with normal pyeloureteric junctions in all. Tissue RNA was chemically extracted after freezing the biopsy samples in liquid nitrogen, with cDNA synthesis performed immediately after nucleic acid isolation. qPCR was performed to evaluate the relative expression of Tgfb1, Mmp1, Timp1, Pai1, Ctgf, and Vegfa. Expression levels of the Gapdh and Gpi genes (geometric average) were used to calculate the relative expression of the investigated genes. Outliers were removed prior to calculating confidence intervals for the experimental groups, and a Wilcoxon rank-sum test was performed to determine the statistical significance of the differences. Results: Significant differences between healthy and stenotic tissue samples in Ctgf gene expression levels were observed, with the samples from afflicted tissue showing lower expression. No statistical difference in expression levels of Tgfb1, Timp1, Vegfa, Mmp1, and Pai1 was found. Conclusions: These findings suggest that tissue fibrosis, similar to other tissues and organs, is not the leading cause of stenosis, at least at the moment of surgery. Decreased CTGF expression is indicative of the developmental origin of obstruction.

Renal fibrosis (RF) is a common pathway leading to chronic kidney disease (CKD), which lacks effective treatment. While estrogen receptor beta (ERβ) is known to be present in the kidney, its role in RF remains unclear. The present study aimed to investigate the role and underlying mechanism of ERβ during RF progression in patients and animal models with CKD. We found that ERβ was highly expressed in the proximal tubular epithelial cells (PTECs) in healthy kidneys but its expression was largely lost in patients with immunoglobin A nephropathy (IgAN) and in mice with unilateral ureter obstruction (UUO) and subtotal nephrectomy (5/6Nx). ERβ deficiency markedly exacerbated, whereas ERβ activation by WAY200070 and DPN attenuated RF in both UUO and 5/6Nx mouse models, suggesting a protective role of ERβ in RF. In addition, ERβ activation inhibited TGF-β1/Smad3 signaling, while loss of renal ERβ was associated with overactivation of the TGF-β1/Smad3 pathway. Furthermore, deletion or pharmacological inhibition of Smad3 prevented the loss of ERβ and RF. Mechanistically, activation of ERβ competitively inhibited the association of Smad3 with the Smad-binding element, thereby downregulating the transcription of the fibrosis-related genes without altering Smad3 phosphorylation in vivo and in vitro. In conclusion, ERβ exerts a renoprotective role in CKD by blocking the Smad3 signaling pathway. Thus, ERβ may represent as a promising therapeutic agent for RF., Competing Interests: Declaration of competing interest The authors declare no disclosures of interests., (Copyright © 2023. Published by Elsevier B.V.)

Chronic kidney disease (CKD) is a major health problem. Kidney fibrosis is a hallmark and final common pathway of CKD. The Hippo/yes-associated protein (YAP) pathway regulates organ size, inflammation, and tumorigenesis. Our previous study demonstrated tubular YAP activation by tubule-specific double knockout of mammalian STE20-like protein kinase 1/2 (Mst1/2) induced CKD in mice, but the underlying mechanisms remain to be fully elucidated. Activator protein (AP)-1 activation was found to promote tubular atrophy and tubulointerstitial fibrosis. Therefore, we studied whether YAP regulates AP-1 expression in the kidney. We found that expression of various AP-1 components was induced in kidneys subjected to unilateral ureteric obstruction and in Mst1/2 double knockout kidneys, and these inductions were blocked by deletion of Yap in tubular cells, with Fosl1 being most affected compared with other AP-1 genes. Inhibition of Yap also most highly suppressed Fosl1 expression among AP-1 genes in HK-2 and IMCD3 renal tubular cells. YAP bound to the Fosl1 promoter and promoted Fosl1 promoter-luciferase activity. Our results suggest that YAP controls AP-1 expression and that Fosl1 is the primary target of YAP in renal tubular cells. NEW & NOTEWORTHY Yes-associated protein (YAP) activation leads to tubular injury, renal inflammation, and fibrosis, but the underlying mechanisms are not fully understood. We now provide genetic evidence that YAP promotes activator protein-1 expression and that Fosl1 is the primary target of YAP in renal tubular cells.

Significance Statement: Congenital obstructive uropathy (COU) is a prevalent human developmental defect with highly heterogeneous clinical presentations and outcomes. Genetics may refine diagnosis, prognosis, and treatment, but the genomic architecture of COU is largely unknown. Comprehensive genomic screening study of 733 cases with three distinct COU subphenotypes revealed disease etiology in 10.0% of them. We detected no significant differences in the overall diagnostic yield among COU subphenotypes, with characteristic variable expressivity of several mutant genes. Our findings therefore may legitimize a genetic first diagnostic approach for COU, especially when burdening clinical and imaging characterization is not complete or available., Background: Congenital obstructive uropathy (COU) is a common cause of developmental defects of the urinary tract, with heterogeneous clinical presentation and outcome. Genetic analysis has the potential to elucidate the underlying diagnosis and help risk stratification., Methods: We performed a comprehensive genomic screen of 733 independent COU cases, which consisted of individuals with ureteropelvic junction obstruction ( n =321), ureterovesical junction obstruction/congenital megaureter ( n =178), and COU not otherwise specified (COU-NOS; n =234)., Results: We identified pathogenic single nucleotide variants (SNVs) in 53 (7.2%) cases and genomic disorders (GDs) in 23 (3.1%) cases. We detected no significant differences in the overall diagnostic yield between COU sub-phenotypes, and pathogenic SNVs in several genes were associated to any of the three categories. Hence, although COU may appear phenotypically heterogeneous, COU phenotypes are likely to share common molecular bases. On the other hand, mutations in TNXB were more often identified in COU-NOS cases, demonstrating the diagnostic challenge in discriminating COU from hydronephrosis secondary to vesicoureteral reflux, particularly when diagnostic imaging is incomplete. Pathogenic SNVs in only six genes were found in more than one individual, supporting high genetic heterogeneity. Finally, convergence between data on SNVs and GDs suggest MYH11 as a dosage-sensitive gene possibly correlating with severity of COU., Conclusions: We established a genomic diagnosis in 10.0% of COU individuals. The findings underscore the urgent need to identify novel genetic susceptibility factors to COU to better define the natural history of the remaining 90% of cases without a molecular diagnosis., (Copyright © 2023 by the American Society of Nephrology.)

DNA-binding protein A (DbpA) belongs to the Y-box family of cold shock domain proteins that exert transcriptional and translational activities in the cell via their ability to bind and regulate mRNA. To investigate the role of DbpA in kidney disease, we utilized the murine unilateral ureter obstruction (UUO) model, which recapitulates many features of obstructive nephropathy seen in humans. We observed that DbpA protein expression is induced within the renal interstitium following disease induction. Compared with wild-type animals, obstructed kidneys from Ybx3 -deficient mice are protected from tissue injury, with a significant reduction in the number of infiltrating immune cells as well as in extracellular matrix deposition. RNAseq data from UUO kidneys show that Ybx3 is expressed by activated fibroblasts, which reside within the renal interstitium. Our data support a role for DbpA in orchestrating renal fibrosis and suggest that strategies targeting DbpA may be a therapeutic option to slow disease progression.

Background: Renal tubulointerstitial fibrosis is the hallmark of various chronic kidney diseases. Symmetric dimethylarginine (SDMA) is an independent cardiovascular risk factor in patients with chronic kidney diseases, which is mostly excreted through renal tubules. However, the effect of SDMA on kidneys in a pathological condition is currently unknown. In this study, we investigated the role of SDMA in renal tubulointerstitial fibrosis and explored its underlying mechanisms., Methods: Mouse unilateral ureteral obstruction (UUO) and unilateral ischemia-reperfusion injury (UIRI) models were established to study renal tubulointerstitial fibrosis. SDMA was injected into kidneys through ureter retrogradely. TGF-β stimulated human renal epithelial (HK2) cells were used as an in vitro model and treated with SDMA. Signal transducer and activator of transcription-4 (STAT4) was inhibited by berbamine dihydrochloride or siRNA or overexpressed by plasmids in vitro. Masson staining and Western blotting were performed to evaluate renal fibrosis. Quantitative PCR was performed to validate findings derived from RNA sequencing analysis., Results: We observed that SDMA (from 0.01 to 10 µM) dose-dependently inhibited the expression of pro-fibrotic markers in TGF-β stimulated HK2 cells. Intrarenal administration of SDMA (2.5 µmol/kg or 25 µmol/kg) dose-dependently attenuated renal fibrosis in UUO kidneys. A significant increase in SDMA concentration (from 19.5 to 117.7 nmol/g, p < 0.001) in mouse kidneys was observed after renal injection which was assessed by LC-MS/MS. We further showed that intrarenal administration of SDMA attenuated renal fibrosis in UIRI induced mouse fibrotic kidneys. Through RNA sequencing analysis, we found that the expression of STAT4 was reduced by SDMA in UUO kidneys, which was further confirmed by quantitative PCR and Western blotting analysis in mouse fibrotic kidneys and renal cells. Inhibition of STAT4 by berbamine dihydrochloride (0.3 mg/ml or 3.3 mg/ml) or siRNA reduced the expression of pro-fibrotic markers in TGF-β stimulated HK2 cells. Furthermore, blockage of STAT4 attenuated the anti-fibrotic effect of SDMA in TGF-β stimulated HK2 cells. Conversely, overexpression of STAT4 reversed the anti-fibrotic effect of SDMA in TGF-β stimulated HK2 cells., Conclusion: Taken together, our study indicates that renal SDMA ameliorates renal tubulointerstitial fibrosis through inhibition of STAT4., (© 2023. The Author(s).)

Progressive fibrosis is a hallmark of chronic kidney disease, but we lack effective treatments to halt this destructive process. Micropeptides (peptides of no more than 100 amino acids) encoded by small open reading frames represent a new class of eukaryotic regulators. Here, we describe that the micropeptide regulator of β-oxidation (MOXI) regulates kidney fibrosis. MOXI expression was found to be up-regulated in human fibrotic kidney disease, and this correlated with the degree of fibrosis and loss of kidney function. MOXI was expressed in the cytoplasm and mitochondria of cultured tubular epithelial cells and translocated to the nucleus upon Transforming Growth Factor-β1 stimulation. Deletion of Moxi protected mice against fibrosis and inflammation in the folic acid and unilateral ureteral obstruction models. As a potential molecular therapy, treatment with an antisense MOXI oligonucleotide effectively knocked-down MOXI expression and protected against kidney fibrosis in both models. Bimolecular fluorescence complementation identified the enzyme N-acetyltransferase 14 (Nat14) and transcription factor c-Jun as MOXI binding partners. The MOXI/Nat14/c-Jun complex enhances basal and Transforming Growth Factor-β1 induced collagen I gene promoter activity. Phosphorylation at T49 is required for MOXI nuclear localization and for complex formation with Nat14 and c-Jun. Furthermore, mice with a MoxiT49A point mutation were protected in the models of kidney fibrosis. Thus, our studies demonstrate a key role for the micropeptide MOXI in kidney fibrosis and identify a new function of MOXI in forming a transcriptional complex with Nat14 and c-Jun., (Copyright © 2023 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Cannabidiol (CBD) is thought to have multiple biological effects, including the ability to attenuate inflammatory processes. Cannabigerols (CBGA and its decarboxylated CBG molecule) have pharmacological profiles similar to CBD. The endocannabinoid system has recently emerged to contribute to kidney disease, however, the therapeutic properties of cannabinoids in kidney disease remain largely unknown. In this study, we determined whether CBD and CBGA can attenuate kidney damage in an acute kidney disease model induced by the chemotherapeutic cisplatin. In addition, we evaluated the anti-fibrosis effects of these cannabinoids in a chronic kidney disease model induced by unilateral ureteral obstruction (UUO). We find that CBGA, but not CBD, protects the kidney from cisplatin-induced nephrotoxicity. CBGA also strongly suppressed mRNA of inflammatory cytokines in cisplatin-induced nephropathy, whereas CBD treatment was only partially effective. Furthermore, both CBGA and CBD treatment significantly reduced apoptosis through inhibition of caspase-3 activity. In UUO kidneys, both CBGA and CBD strongly reduced renal fibrosis. Finally, we find that CBGA, but not CBD, has a potent inhibitory effect on the channel-kinase TRPM7. We conclude that CBGA and CBD possess reno-protective properties, with CBGA having a higher efficacy, likely due to its dual anti-inflammatory and anti-fibrotic effects paired with TRPM7 inhibition., (© 2023. The Author(s).)

Planar cell polarity (PCP), a process of coordinated alignment of cell polarity across the tissue plane, may contribute to the repair of renal tubules after kidney injury. Intu is a key effector protein of PCP. Herein, conditional knockout (KO) mouse models that ablate Intu specifically from kidney tubules (Intu KO) were established. Intu KO mice and wild-type littermates were subjected to unilateral renal ischemia/reperfusion injury (IRI) or unilateral ureteral obstruction. Kidney repair was evaluated by histologic, biochemical, and immunohistochemical analyses. In vitro, scratch wound healing was examined in Intu-knockdown and control renal tubular cells. Ablation of Intu in renal tubules delayed kidney repair and ameliorated renal fibrosis after renal IRI. Intu KO mice had less renal fibrosis during unilateral ureteral obstruction. Mechanistically, Intu KO kidneys had less senescence but higher levels of cell proliferation and apoptosis during kidney repair after renal IRI. In vitro, Intu knockdown suppressed scratch wound healing in renal tubular cells, accompanied by the abnormality of centrosome orientation. Together, the results provide the first evidence for the involvement of PCP in tubular repair after kidney injury, shedding light on new strategies for improving kidney repair and recovery., (Copyright © 2023 American Society for Investigative Pathology. All rights reserved.)

Severe hydronephrosis increases the risk of urinary tract infection and irretrievable renal fibrosis. While TGFβ1-mediated fibrotic changes in proximal tubular epithelial cells and fatty acid oxidation (FAO) deregulation contribute to renal fibrosis and hydronephrosis. Firstly, a few elements were analyzed in this paper, including differentially-expressed long non-coding RNAs (lncRNAs), and miRNAs correlated to CPT1A, RXRA, and NCOA1. This paper investigated TGFβ1 effects on lncRNA FABP5P3, CPT1A, RXRA, and NCOA1 expression and fibrotic changes in HK-2 cells and FABP5P3 overexpression effects on TGFβ1-induced changes. Moreover, this paper predicted and proved that miR-22 binding to lncRNA FABP5P3, 3'UTR of CPT1A, RXRA, and NCOA1 was validated. The dynamic effects of the FABP5P3/miR-22 axis on TGFβ1-induced changes were investigated. A Renal fibrosis model was established in unilateral ureteral obstruction (UUO) mice, and FABP5P3 effects were investigated. Eventually, this paper concluded that TGFβ1 inhibited lncRNA FABP5P3, CPT1A, RXRA, and NCOA1 expression, induced fibrotic changes in HK-2 cells, and induced metabolic reprogramming within HK-2 cells, especially lower FAO. FABP5P3 overexpression partially reversed TGFβ1-induced changes. miR-22 targeted lncRNA FABP5P3, CPT1A, RXRA, and NCOA1. LncRNA FABP5P3 counteracted miR-22 inhibition of CPT1A, NCOA1, and RXRA through competitive binding. TGFβ1 stimulation induced the activation of TGFβ/SMAD and JAG/Notch signaling pathways; Nocth2 knockdown reversed TGFβ1 suppression on lncRNA FABP5P3. FABP5P3 overexpression attenuated renal fibrosis in unilateral ureteral obstruction mice. The LncRNA FABP5P3/miR-22 axis might be a potent target for improving the FAO deregulation and fibrotic changes in proximal TECs under TGFβ1 stimulation.

Tubulointerstitial fibrosis (TIF) is essential during the development of end-stage kidney disease (ESKD) and is associated with the impairment of fatty acid oxidation (FAO). Kruppel-like factor 14 (KLF14) is an important gene in lipid metabolism, but its role in TIF remains unknown. TGF-β-stimulated HK-2 cells and mouse unilateral ureteral obstruction (UUO) were used as renal fibrosis models. The role of KLF14 in the process of renal fibrosis was verified by gene knockout mice, genetic or pharmacological interference in animal model and cell model respectively. In the current study, we found that KLF14 expression increased after activation of the TGF-β signaling pathway during TIF. In KLF14 -/- mice, more severe fibrosis was observed after unilateral ureteral obstruction (UUO) was induced. In human HK2 cells, knockdown of KLF14 led to more severe fibrosis induced by TGF-β1, while overexpression of KLF14 partially attenuated this process. Specifically, KLF14 deficiency decreased mitochondrial FAO activity, resulting in lipid accumulation. Thus, the energy supply to the cells was insufficient, finally resulting in TIF. We further proved that KLF14 could target peroxisome proliferator activated receptor alpha (PPARα) as a transcriptional activator. This study identified the upregulation of KLF14 expression in response to kidney stress during the process of fibrosis. Upon TIF, the activated TGF-β signaling pathway can enhance KLF14 expression, while the upregulation of KLF14 expression can decrease the degree of TIF by improving FAO activity in tubular epithelial cells and recovering the energy supply mediated by PPARα., Competing Interests: Declaration of competing interest The authors declare no competing financial interests., (Copyright © 2022. Published by Elsevier Inc.)

Renal fibrosis is a major factor in the progression of chronic kidney diseases. Obstructive nephropathy is a common cause of renal fibrosis, which is also accompanied by inflammation. To explore the effect of human-specific CHRFAM7A expression, an inflammation-related gene, on renal fibrosis during obstructive nephropathy, we studied CHRFAM7A transgenic mice and wild type mice that underwent unilateral ureteral obstruction (UUO) injury. Transgenic overexpression of CHRFAM7A gene inhibited UUO-induced renal fibrosis, which was demonstrated by decreased fibrotic gene expression and collagen deposition. Furthermore, kidneys from transgenic mice had reduced TGF-β1 and Smad2/3 expression following UUO compared with those from wild type mice with UUO. In addition, the overexpression of CHRFAM7A decreased release of inflammatory cytokines in the kidneys of UUO-injured mice. In vitro, the overexpression of CHRFAM7A inhibited TGF-β1-induced increase in expression of fibrosis-related genes in human renal tubular epithelial cells (HK-2 cells). Additionally, up-regulated expression of CHRFAM7A in HK-2 cells decreased TGF-β1-induced epithelial-mesenchymal transition (EMT) and inhibited activation f TGF-β1/Smad2/3 signalling pathways. Collectively, our findings demonstrate that overexpression of the human-specific CHRFAM7A gene can reduce UUO-induced renal fibrosis by inhibiting TGF-β1/Smad2/3 signalling pathway to reduce inflammatory reactions and EMT of renal tubular epithelial cells., (© 2022 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

The role of exogenous microRNAs (miRNAs) in renal fibrosis is poorly understood. Here, the effect of exogenous miRNAs on renal fibrosis was investigated using a renal fibrosis mouse model generated by unilateral ureteral obstruction (UUO). miRNA microarray analysis and quantitative reverse-transcription polymerase chain reaction showed that miR-122-5p was the most downregulated (0.28-fold) miRNA in the kidneys of UUO mice. The injection of an miR-122-5p mimic promoted renal fibrosis and upregulated COL1A2 and FN1 , whereas an miR-122-5p inhibitor suppressed renal fibrosis and downregulated COL1A2 and FN1 . The expression levels of fibrosis-related mRNAs, which were predicted targets of miR-122-5p, were evaluated. The expression level of TGFBR2 , a pro-fibrotic mRNA, was upregulated by the miR-122-5p mimic, and the expression level of FOXO3 , an anti-fibrotic mRNA, was upregulated by the miR-122-5p inhibitor. The protein expressions of TGFBR2 and FOXO3 were confirmed by immunohistochemistry. Additionally, the expression levels of LC3 , downstream anti-fibrotic mRNAs of FOXO3 , were upregulated by the miR-122-5p inhibitor. These results suggest that miR-122-5p has critical roles in renal fibrosis.

Background: A novel collagen called type XXVIII collagen (COL28) is involved in cancer and lung fibrosis. Preliminary data showed that renal tubular epithelial cells could proliferate, migrate, and undergo an epithelial-mesenchymal transition (EMT) when COL28 was overexpressed; however, it is still unknown how this occurs and what the underlying mechanism is., Methods: We analyzed the differential expression of genes (DEGs) in the stable COL28 overexpression HK-2 cell lines by RNA-sequencing analysis, before which Gene Ontology (GO) and Kyoto Encyclopedia of Gene and Genomes (KEGG) analyses were performed. Genes related to COL28 promoting HK-2 cell proliferation and EMT were screened and verified. By using western blot and immunofluorescence, the effects of COL28 on the expression of α -SMA, E-cadherin, Snail, HKDC1, and SREBP1 were detected. The effect of COL28 overexpression on renal fibrosis in unilateral ureteral obstruction (UUO) mice was detected by H&E and Masson staining. HKDC1 interference agent was synthesized and transfected into the HK-2 cell line stably overexpressing COL28. In HK-2 cells, the effects of HKDC1 interference on the expression of α -SMA, E-cadherin, and Snail were detected., Results: We screened and verified that HKDC1 was related to COL28 and promoted HK-2 cell proliferation and EMT. WB showed that in HK-2 cells, COL28 overexpression increased α -SMA, Snail, HKDC1, and SREBP1 expressions and decreased E-cadherin expression. Overexpression of COL28 aggravated renal interstitial fibrosis in UUO mice; upregulated α -SMA, Snail, HKDC1, and SREBP1 expressions; and decreased the E-cadherin protein expression in UUO mice. Interference of HKDC1 expression promoted the E-cadherin protein expression while inhibiting α -SMA, Snail, HKDC1, and SREBP1 protein expressions., Conclusion: Overexpression of COL28 can aggravate renal interstitial fibrosis by encouraging renal tubular epithelial cells to undergo EMT, and interference with HKDC1 expression can alleviate fibrosis by reversing EMT induced by COL28 overexpression., Competing Interests: No conflicts of interest are disclosed by the authors., (Copyright © 2022 Linlin Li et al.)

Fibrosis is a relentlessly progressive and irreversible cause of organ damage, as in chronic kidney disease (CKD), but its underlying mechanisms remain elusive. We found that a circular RNA, circPTPN14, is highly expressed in human kidneys with biopsy-proved chronic interstitial fibrosis, mouse kidneys subjected to ischemia/reperfusion (IR) or unilateral ureteral obstruction (UUO), and TGFβ1-stimulated renal tubule epithelial cells (TECs). The intrarenal injection of circPTPN14 shRNA alleviated the progression of fibrosis in kidneys subjected to IR or UUO. Knockdown of circPTPN14 in TECs inhibited TGFβ1-induced expression of profibrotic genes, whereas overexpressing circPTPN14 increased the profibrotic effect of TGFβ1. The profibrotic action of circPTPN14 was ascribed to an increase in MYC transcription. The binding of circPTPN14 to the KH3 and KH4 domains of far upstream element (FUSE) binding protein 1 (FUBP1) enhanced the interaction between FUBP1 and FUSE domain, which was required for the initiation of MYC transcription. In human kidneys (n = 30) with biopsy-proved chronic interstitial fibrosis, the expression of circPTPN14 positively correlated with MYC expression. Taken together these studies show a novel mechanism in the pathogenesis of renal fibrosis, mediated by circPTPN14, which can be a target in the diagnosis and treatment of CKD., (© 2022. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Renal fibrosis, the final pathway of chronic kidney disease, is caused by genetic and epigenetic mechanisms. Although DNA methylation has drawn attention as a developing mechanism of renal fibrosis, its contribution to renal fibrosis has not been clarified. To address this issue, the effect of zebularine, a DNA methyltransferase inhibitor, on renal inflammation and fibrosis in the murine unilateral ureteral obstruction (UUO) model was analyzed. Zebularine significantly attenuated renal tubulointerstitial fibrosis and inflammation. Zebularine decreased trichrome, α-smooth muscle actin, collagen IV, and transforming growth factor-β1 staining by 56.2%. 21.3%, 30.3%, and 29.9%, respectively, at 3 days, and by 54.6%, 41.9%, 45.9%, and 61.7%, respectively, at 7 days after UUO. Zebularine downregulated mRNA expression levels of matrix metalloproteinase (MMP)-2, MMP-9, fibronectin, and Snail1 by 48.6%. 71.4%, 31.8%, and 42.4%, respectively, at 7 days after UUO. Zebularine also suppressed the activation of nuclear factor-κB (NF-κB) and the expression of pro-inflammatory cytokines, including tumor necrosis factor-α, interleukin (IL)-1β, and IL-6, by 69.8%, 74.9%, and 69.6%, respectively, in obstructed kidneys. Furthermore, inhibiting DNA methyltransferase buttressed the nuclear expression of nuclear factor (erythroid-derived 2)-like factor 2, which upregulated downstream effectors such as catalase (1.838-fold increase at 7 days, p < 0.01), superoxide dismutase 1 (1.494-fold increase at 7 days, p < 0.05), and NAD(P)H: quinone oxidoreduate-1 (1.376-fold increase at 7 days, p < 0.05) in obstructed kidneys. Collectively, these findings suggest that inhibiting DNA methylation restores the disrupted balance between pro-inflammatory and anti-inflammatory pathways to alleviate renal inflammation and fibrosis. Therefore, these results highlight the possibility of DNA methyltransferases as therapeutic targets for treating renal inflammation and fibrosis.

Background: Renal interstitial fibrosis (RIF) is the main pathological change of a variety of chronic kidney diseases (CKD). Epigenetic modifications of fibrosis-prone genes regulate RIF progression. This study aimed to investigate long non-coding RNA (lncRNA) N6-methyladenosine (m 6 A) modification and its role in regulating RIF progression., Methods: Unilateral ureteral occlusion (UUO) was employed to construct the RIF in vivo model; and TGF-β1-treated HK-2 and HKC-8 cells were used for in vitro experiments. The mRNA and protein expressions were assessed using qRT-PCR and western blot. The proliferation and migration were evaluated by EdU assay and transwell assay, respectively. In addition, levels of inflammatory cytokines were determined by ELISA assay and qRT-PCR. Moreover, lncRNA GAS5 m 6 A level was detected using Me-RIP assay. HE and Masson staining were employed to evaluate fibrotic lesions of the kidney., Results: FTO expression was elevated in HK-2 and HKC-8 cells after TGF-β1 treatment and mouse kidney tissue following UUO, and lncRNA GAS5 was downregulated. LncRNA GAS5 overexpression or FTO silencing suppressed TGF-β1-induced the increase of EMT-related proteins (Vimentin, Snail and N-cadherin) and inflammatory cytokines (IL-6, IL-1β and TNF-α) levels in HK-2 cells. FTO suppressed lncRNA GAS5 expression by reducing the m6A modification of lncRNA GAS5. Additionally, FTO knockdown could suppress EMT process and inflammation response induced by TGF-β1 and UUO in vitro and in vivo. As expected, FTO knockdown abrogated the promotion effects of lncRNA GAS5 silencing on TGF-β1-induced EMT process and inflammation response in HK-2 and HKC-8 cells., Conclusion: FTO promoted EMT process and inflammation response through reducing the m 6 A modification of lncRNA GAS5., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

The global burden of chronic kidney disease is increasing, and the majority of these diseases are progressive. Special site-targeted drugs are emerging as alternatives to traditional drugs. Oligonucleotides (ODNs) have been proposed as effective therapeutic tools in specific molecular target therapies for several diseases. We designed ring-type non-coding RNAs (ncRNAs), also called mTOR ODNs to suppress mammalian target rapamycin (mTOR) translation. mTOR signaling is associated with excessive cell proliferation and fibrogenesis. In this study, we examined the effects of mTOR suppression on chronic renal injury. To explore the regulation of fibrosis and inflammation in unilateral ureteral obstruction (UUO)-induced injury, we injected synthesized ODNs via the tail vein of mice. The expression of inflammatory-related markers (interleukin-1β, tumor necrosis factor-α), and that of fibrosis (α-smooth muscle actin, fibronectin), was decreased by synthetic ODNs. Additionally, ODN administration inhibited the expression of autophagy-related markers, microtubule-associated protein light chain 3, Beclin1, and autophagy-related gene 5-12. We confirmed that ring-type ODNs inhibited fibrosis, inflammation, and autophagy in a UUO mouse model. These results suggest that mTOR may be involved in the regulation of autophagy and fibrosis and that regulating mTOR signaling may be a therapeutic strategy against chronic renal injury.

Acidic nuclear phosphoprotein 32 family member e (Anp32e) has been reported to contribute to early mammalian development and cancer metastasis. However, the pathophysiological role of Anp32e in renal interstitial fibrosis (RIF) is poorly understood. Here, we demonstrated that Anp32e was highly expressed in the region of RIF in patients with IgA nephropathy, unilateral ureteral obstruction (UUO) mouse kidneys, and Boston University mouse proximal tubular (BUMPT) cells when treated with TGF-β1; this upregulation was positively correlated with the total fibrotic area of the kidneys. The overexpression of Anp32e enhanced the TGF-β1-induced production of fibrosis-related proteins (fibronectin (Fn) and collagen type I (Col-I)) in BUMPT cells whereas the knockdown of Anp32e suppressed the deposition of these fibrosis-related proteins in UUO mice and TGF-β1-stimulated BUMPT cells. In particular, Anp32e overexpression alone induced the deposition of Fn and Col-I in both mouse kidneys and BUMPT cells without TGF-β1 stimulation. Furthermore, we revealed that the overexpression of Anp32e induced the expression of TGF-β1 and p-Smad3 while TGF-β1 inhibitor SB431542 reversed the Anp32e-induced upregulation of Fn and Col-I in BUMPT cells without TGF-β1 stimulation. Collectively, our data demonstrate that Anp32e promotes the deposition of fibrosis-related proteins by regulating the TGF-β1/Smad3 pathway., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Introduction: Cardiovascular disease constitutes the leading cause of mortality in patients with chronic kidney disease (CKD), which is termed cardiorenal syndrome type 4 (CRS-4). Here, we report the development of pathological cardiac remodeling and fibrosis in unilateral urinary obstruction (UUO) rats., Methods: Hematoxylin and eosin (H&E) staining was performed to observe the pathology of myocardial tissue. The degree of myocardial tissue fibrosis was observed by Masson and Sirius red staining. Immunohistochemical staining was applied to detect the expression of CD34 and CD105 in myocardial tissue, and immunofluorescent staining was performed to examine the expression of CD34, collagen I/collagen III, and alpha smooth muscle actin ( α -SMA). The expression of the signal pathway-related proteins vascular endothelial growth factor A (VEGFA), vascular endothelial growth factor receptor 2 (VEGFR2), nuclear factor κ B (NF- κ B), and interleukin (IL)-1 β was tested by western blotting. Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the mRNA levels of serum and glucocorticoid-inducible kinase (SGK)-1, NF- κ B, and interleukin-1 β (IL-1 β )., Results: The results showed the development of pathological cardiac remodeling and cardiac dysfunction in UUO rats. Moreover, there was more angiogenesis and endothelial-mesenchymal transition (End-MT) in the UUO group, and these effects were inhibited by eplerenone., Conclusions: The results indicated that this cardiac fibrosis was associated with angiogenesis and that End-MT was related to aldosterone and mineralocorticoid receptor (MR) activation. Moreover, in association with the MR/IL-1 β /VEGFA signaling pathway, early treatment with the MR antagonist eplerenone in rats with UUO-induced CKD may significantly attenuate MR activation and cardiac fibrosis., Competing Interests: The authors have no conflicts of interest to declare., (Copyright © 2022 Yi Chang et al.)

Clopidogrel, a P2Y12 inhibitor, is a novel anti-fibrosis agent for chronic kidney disease (CKD), but its mechanisms remain unclear, which we investigated by silencing P2Y12 or treating unilateral ureteral obstruction (UUO) in LysM-Cre/Rosa Tomato mice with clopidogrel in vivo and in vitro. We found that P2Y12 was significantly increased and correlated with progressive renal fibrosis in CKD patients and UUO mice. Phenotypically, up to 82% of P2Y12-expressing cells within the fibrosing kidney were of macrophage origin, identified by co-expressing CD68/F4/80 antigens or a macrophage-lineage-tracing marker Tomato. Unexpectedly, more than 90% of P2Y12-expressing macrophages were undergoing macrophage-to-myofibroblast transition (MMT) by co-expressing alpha smooth muscle actin (α-SMA), which was also confirmed by single-cell RNA sequencing. Functionally, clopidogrel improved the decline rate of the estimated glomerular filtration rate (eGFR) in patients with CKD and significantly inhibited renal fibrosis in UUO mice. Mechanistically, P2Y12 expression was induced by transforming growth factor β1 (TGF-β1) and promoted MMT via the Smad3-dependent mechanism. Thus, silencing or pharmacological inhibition of P2Y12 was capable of inhibiting TGF-β/Smad3-mediated MMT and progressive renal fibrosis in vivo and in vitro. In conclusion, P2Y12 is highly expressed by macrophages in fibrosing kidneys and mediates renal fibrosis by promoting MMT via TGF-β/Smad3 signaling. Thus, P2Y12 inhibitor maybe a novel and effective anti-fibrosis agent for CKD., Competing Interests: Declaration of interests All the authors declared no competing interests., (Copyright © 2022 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

Renal tubulointerstitial fibrosis is the hallmark of chronic kidney disease (CKD) and the best predictor of renal survival. However, current treatments for CKD remain extremely limited. Therefore, novel therapeutic targets are urgently needed to either stop or reverse CKD progression. The present study was designed to explore the potential role of GPR87, a member of the G protein-coupled receptors (GPCRs) family, in the pathogenesis of tubulointerstitial fibrosis. It was found that GPR87 was significantly induced in the kidney, especially in tubular areas, from different mouse models of renal fibrosis, including unilateral ureteral obstruction (UUO) nephropathy, aristolochic acid nephropathy, and diabetic nephropathy, respectively. Tubule-specific GPR87 deletion dramatically ameliorated tubulointerstitial fibrosis in UUO mice. Mechanistically, GPR87 accelerated glycolysis and mitochondrial injury by YAP-hexokinase-2 signaling, thereby promoting renal fibrosis. Importantly, the upregulation of GPR87 was also found in the kidney from patients with various CKD, indicating that the induction of GPR87 may be a common feature of human kidney diseases. Collectively, our studies for the first time demonstrate that GPR87 plays a pivotal role in renal fibrosis at least in part by accelerating glycolysis and mitochondrial injury, suggesting that targeting GPR87 may represent a novel therapeutic strategy for patients with CKD., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Renal fibrosis is a common consequence of various progressive nephropathies, including obstructive nephropathy, and ultimately leads to kidney failure. Infiltration of inflammatory cells is a prominent feature of renal injury after draining blockages from the kidney, and correlates closely with the development of renal fibrosis. However, the underlying molecular mechanism behind the promotion of renal fibrosis by inflammatory cells remains unclear. Herein, we showed that unilateral ureteral obstruction (UUO) induced Gasdermin D (GSDMD) activation in neutrophils, abundant neutrophil extracellular traps (NETs) formation and macrophage-to-myofibroblast transition (MMT) characterized by α-smooth muscle actin (α-SMA) expression in macrophages. Gsdmd deletion significantly reduced infiltration of inflammatory cells in the kidneys and inhibited NETs formation, MMT and thereby renal fibrosis. Chimera studies confirmed that Gsdmd deletion in bone marrow-derived cells, instead of renal parenchymal cells, provided protection against renal fibrosis. Further, specific deletion of Gsdmd in neutrophils instead of macrophages protected the kidney from undergoing fibrosis after UUO. Single-cell RNA sequencing identified robust crosstalk between neutrophils and macrophages. In vitro, GSDMD-dependent NETs triggered p65 translocation to the nucleus, which boosted the production of inflammatory cytokines and α-SMA expression in macrophages by activating TGF-β1/Smad pathway. In addition, we demonstrated that caspase-11, that could cleave GSDMD, was required for NETs formation and renal fibrosis after UUO. Collectively, our findings demonstrate that caspase-11/GSDMD-dependent NETs promote renal fibrosis by facilitating inflammation and MMT, therefore highlighting the role and mechanisms of NETs in renal fibrosis., (© 2022. The Author(s).)

Background: Renal fibrosis is a serious condition that results in the development of chronic kidney diseases. The MEN1 gene is an epigenetic regulator that encodes the menin protein and its role in kidney tissue remains unclear., Methods: Kidney histology was examined on paraffin sections stained with hematoxylin-eosin staining. Masson's trichrome staining and Sirius red staining were used to analyze renal fibrosis. Gene and protein expression were determined by quantitative real-time PCR (qPCR) and Western blot, respectively. Immunohistochemistry staining in the kidney tissues from mice or patients was used to evaluate protein levels. Flow cytometry was used to analyze the cell cycle distributions and apoptosis. RNA-sequencing was performed for differential expression genes in the kidney tissues of the Men1f/f and Men1∆/∆ mice. Chromatin immunoprecipitation sequencing (ChIP-seq) was carried out for identification of menin- and H3K4me3-enriched regions within the whole genome in the mouse kidney tissue. ChIP-qPCR assays were performed for occupancy of menin and H3K4me3 at the gene promoter regions. Luciferase reporter assay was used to detect the promoter activity. The exacerbated unilateral ureteral obstruction (UUO) models in the Men1f/f and Men1∆/∆ mice were used to assess the pharmacological effects of rh-HGF on renal fibrosis., Results: The expression of MEN1 is reduce in kidney tissues of fibrotic mouse and human diabetic patients and treatment with fibrotic factor results in the downregulation of MEN1 expression in renal tubular epithelial cells (RTECs). Disruption of MEN1 in RTECs leads to high expression of α-SMA and Collagen 1, whereas MEN1 overexpression restrains epithelial-to-mesenchymal transition (EMT) induced by TGF-β treatment. Conditional knockout of MEN1 resulted in chronic renal fibrosis and UUO-induced tubulointerstitial fibrosis (TIF), which is associated with an increased induction of EMT, G2/M arrest and JNK signaling. Mechanistically, menin recruits and increases H3K4me3 at the promoter regions of hepatocyte growth factor (HGF) and a disintegrin and metalloproteinase with thrombospondin motifs 5 (Adamts5) genes and enhances their transcriptional activation. In the UUO mice model, exogenous HGF restored the expression of Adamts5 and ameliorated renal fibrosis induced by Men1 deficiency., Conclusions: These findings demonstrate that MEN1 is an essential antifibrotic factor in renal fibrogenesis and could be a potential target for antifibrotic therapy., (© 2022 The Authors. Clinical and Translational Medicine published by John Wiley & Sons Australia, Ltd on behalf of Shanghai Institute of Clinical Bioinformatics.)

Patients with chronic kidney disease and experimental animal models of kidney fibrosis manifest diverse progression rates. Genetic susceptibility may contribute to this diversity, but the causes remain largely unknown. We have previously described kidney fibrosis with a mild or severe phenotype in mice expressing transforming growth factor-beta1 (TGF-β1) under the control of a mouse albumin promoter (Alb/TGF-β1), on a mixed genetic background with CBAxC57Bl6 mice. Here, we aimed to examine how genetic background may influence kidney fibrosis in TGF-β1 transgenic mice, and in the unilateral ureteral obstruction (UUO) and subtotal nephrectomy (SNX) mouse models. Congenic C57Bl6(B6)-TGFβ and CBAxB6-TGFβ (F1) transgenic mice were generated and survival, proteinuria, kidney histology, transcriptome and protein expressions were analyzed. We investigated the kidneys of B6 and CBA mice subjected to UUO and SNX, and the effects of tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) neutralization on the fibrotic process. CBAxB6-TGFβ mice developed severe kidney fibrosis and premature death, while B6-TGF-β mice had mild fibrosis and prolonged survival. Kidney early growth response factor-2 (EGR2) and TIMP-1 expression were induced only in CBAxB6-TGFβ mice. Similar strain-dependent early changes in EGR2 and TIMP-1 of mice subjected to UUO or SNX were observed. TIMP-1 neutralization in vivo hindered fibrosis both in transgenic mice and the SNX model. EGR2 over-expression in cultured HEK293 cells induced TIMP-1 while EGR2 silencing hindered TGF-β induced TIMP-1 production in HK-2 cells and ureteral obstructed kidneys. Finally, EGR2 and TIMP1 was increased in human kidneys manifesting focal segmental glomerulosclerosis suggesting a correlation between animal studies and patient clinical settings. Thus, our observations demonstrate a strong relationship between genetic background and the progression of kidney fibrosis, which might involve early altered EGR2 and TIMP-1 response, but the relationship to patient genetics remains to be explored., (Copyright © 2022 International Society of Nephrology. All rights reserved.)

Metabolic syndrome is a significant worldwide public health challenge and is inextricably linked to adverse renal and cardiovascular outcomes. The inhibition of the transient receptor potential cation channel subfamily C member 6 (TRPC6) has been found to ameliorate renal outcomes in the unilateral ureteral obstruction (UUO) of accelerated renal fibrosis. Therefore, the pharmacological inhibition of TPRC6 could be a promising therapeutic intervention in the progressive tubulo-interstitial fibrosis in hypertension and metabolic syndrome. In the present study, we hypothesized that the novel selective TRPC6 inhibitor SH045 (larixyl N-methylcarbamate) ameliorates UUO-accelerated renal fibrosis in a New Zealand obese (NZO) mouse model, which is a polygenic model of metabolic syndrome. The in vivo inhibition of TRPC6 by SH045 markedly decreased the mRNA expression of pro-fibrotic markers ( Col1α1 , Col3α1 , Col4α1 , Acta2 , Ccn2 , Fn1 ) and chemokines ( Cxcl1 , Ccl5 , Ccr2 ) in UUO kidneys of NZO mice compared to kidneys of vehicle-treated animals. Renal expressions of intercellular adhesion molecule 1 (ICAM-1) and α-smooth muscle actin (α-SMA) were diminished in SH045- versus vehicle-treated UUO mice. Furthermore, renal inflammatory cell infiltration (F4/80+ and CD4+) and tubulointerstitial fibrosis (Sirius red and fibronectin staining) were ameliorated in SH045-treated NZO mice. We conclude that the pharmacological inhibition of TRPC6 might be a promising antifibrotic therapeutic method to treat progressive tubulo-interstitial fibrosis in hypertension and metabolic syndrome.

The roles of neutrophils in renal inflammation are currently unclear. On examining these cells in the unilateral ureteral obstruction murine model of chronic kidney disease, we found that the injured kidney bore a large and rapidly expanding population of neutrophils that expressed the eosinophil marker Siglec-F. We first verified that these cells were neutrophils. Siglec-F+ neutrophils were recently detected in several studies in other disease contexts. We then showed that a) these cells were derived from conventional neutrophils in the renal vasculature by TGF-β1 and GM-CSF; b) they differed from their parent cells by more frequent hypersegmentation, higher expression of profibrotic inflammatory cytokines, and notably, expression of collagen 1; and c) their depletion reduced collagen deposition and disease progression, but adoptive transfer increased renal fibrosis. These findings have thus unveiled a subtype of neutrophils that participate in renal fibrosis and a potentially new therapeutic target in chronic kidney disease.