ISG15 accelerates acute kidney injury and the subsequent AKI-to-CKD transition by promoting TGFßR1 ISGylation.

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- ß1 (TGF-ß1) 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-ß receptor (TGFßR1). 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-ß1 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 TGFßR1, and inhibited its ubiquitination. Moreover, knockout of TGFßR1 blocked ISG15's fibrosis-exacerbating effect in HK-2 cells, while overexpression of TGFßR1 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 TGFßR1 ISGylation.

Regulation of the RhoA exchange factor GEF-H1 by profibrotic stimuli through a positive feedback loop involving RhoA, MRTF, and Sp1.

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-PK1 tubular cells with tumor necrosis factor (TNF)-α or transforming growth factor (TGF)-ß1 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.

MicroRNA-10 Family Promotes Renal Fibrosis through the VASH-1/Smad3 Pathway.

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-ß1 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-ß1-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.

Huoxue Jiedu Huayu recipe inhibits macrophage-secreted vascular endothelial growth factor-a on angiogenesis and alleviates renal fibrosis in the contralateral kidneys of unilateral ureteral obstruction rats.

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.

Bulk and single-cell transcriptome profiling identify potential cellular targets of the long noncoding RNA Gas5 in renal 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.

LncRNA Meg3 Aggravates Renal Fibrosis Caused by Unilateral Ureteral Obstruction in Rats by Activating the Hedgehog Pathway.

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.

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

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

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

Epigenetic regulations, including DNA methylation, are critical to the development and progression of kidney fibrosis, but the underlying mechanisms remain elusive. Here, we show that fibrosis of the mouse kidney was associated with the induction of DNA methyltransferases and increases in global DNA methylation and was alleviated by the DNA methyltransferase inhibitor 5-Aza-2'-deoxycytidine (5-Aza). Genome-wide analysis demonstrated the hypermethylation of 94 genes in mouse unilateral ureteral obstruction kidneys, which was markedly reduced by 5-Aza. Among these genes, Hoxa5 was hypermethylated at its gene promoter, and this hypermethylation was associated with reduced HOXA5 expression in fibrotic mouse kidneys after ureteral obstruction or unilateral ischemia-reperfusion injury. 5-Aza prevented Hoxa5 hypermethylation, restored HOXA5 expression, and suppressed kidney fibrosis. 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.

Integrative transcriptomic and proteomic profile revealed inhibition of oxidative phosphorylation and peroxisomes during renal interstitial fibrosis.

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.

Progranulin deficiency attenuates tubulointerstitial injury in a mouse unilateral ureteral obstruction model.

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.

Shared and Distinct Renal Transcriptome Signatures in 3 Standard Mouse Models of Chronic Kidney Disease.

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.

HIF-1 contributes to autophagy activation via BNIP3 to facilitate renal fibrosis in hypoxia in vitro and UUO in vivo.

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.

SNHG16/miR-205/HDAC5 is involved in the progression of renal fibrosis.

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.

LncRNA CASC2 Alleviates Renal Interstitial Inflammation and Fibrosis through MEF2C Downregulation-Induced Hinderance of M1 Macrophage Polarization.

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.

Elucidating the role of nicotinamide N-methyltransferase-p53 axis in the progression of chronic kidney disease.

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-ß1 (TGF-ß1), 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.

Pilot Study on the Molecular Pathogenesis of Pyeloureteral Junction Obstruction: Underdevelopment or Fibrosis?

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 TGFß 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.

Knockdown of lncRNA MALAT1 attenuates renal interstitial fibrosis through miR-124-3p/ITGB1 axis.

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-ß1 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-ß1 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.

Tubular cell transcriptional intermediary factor 1γ deficiency exacerbates kidney injury-induced tubular cell polyploidy and fibrosis.

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-ß (TGF-ß)-dependent and -independent signaling. TIF1γ hindered TGF-ß 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-ß 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-ß signaling. Hence, our findings suggest that maintaining homeostasis of tubular TIF1γ may be a new therapeutic option for treating tubulointerstitial fibrosis and subsequent CKD.

Cold Shock Domain Protein DbpA Orchestrates Tubular Cell Damage and Interstitial Fibrosis in Inflammatory Kidney Disease.

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.

Upregulation of miR-200a improves ureteral obstruction-induced renal fibrosis via GAB1/Wnt/ß-catenin signaling.

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/ß-catenin in UUO rats. Moreover, miR-200a expression was inhibited, while GAB1 expression was facilitated in the TGF-ß1-induced HK-2 cells. In TGF-ß1-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-ß1-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/ß-catenin signaling activation, EMT and ECM accumulation. CONCLUSION: Overall, miR-200a increasing improved renal fibrosis through attenuating EMT and ECM accumulation by limiting Wnt/ß-catenin signaling via sponging GAB1, indicating miR-200a may be a promising objective for renal disease therapy.