Exosomal let-7b-5p deriving from parietal epithelial cells attenuate renal fibrosis through suppression of TGFßR1 and ARID3a in obstructive kidney disease.

As renal progenitor cells, parietal epithelial cells (PECs) have demonstrated multilineage differentiation potential in response to kidney injury. However, the function of exosomes derived from PECs has not been extensively explored. Immunofluorescent staining of Claudin-1 was used to identify primary PECs isolated from mouse glomeruli. Transmission electron microscopy, nanoparticle tracking analysis, and western blotting were used to characterize the properties of PECs-derived exosomes (PEC-Exo). The therapeutic role of PEC-Exo in tubulointerstitial fibrosis was investigated in the unilateral ureteral obstruction (UUO) mouse model and TGF-ß1-stimulated HK-2 cells. High-throughput miRNA sequencing was employed to profile PEC-Exo miRNAs. One of the most enriched miRNAs in PEC-Exo was knocked down by transfecting miRNA inhibitor, and then we investigated whether this candidate miRNA was involved in PEC-Exo-mediated tubular repair. The primary PECs expressed Claudin-1, PEC-Exo was homing to obstructed kidney, and TGF-ß1 induced HK-2 cells. PEC-Exo significantly alleviated renal inflammation and ameliorated tubular fibrosis both in vivo and in vitro. Mechanistically, let-7b-5p, highly enriched in PEC-Exo, downregulated the protein levels of transforming growth factor beta receptor 1(TGFßR1) and AT-Rich Interaction Domain 3A(ARID3a) in tubular epithelial cells (TECs), leading to the inhibition of p21 and p27 to restoring cell cycle. Furthermore, administration of let-7b-5p agomir mitigated renal fibrosis in vivo. Our findings demonstrated that PEC-derived exosomes significantly repressed the expression of TGFßR1 and ARID3a by delivering let-7b-5p, thereby alleviating renal fibrosis. This study provides novel insights into the role of PEC-Exo in the repair of kidney injury and new ideas for renal fibrosis intervention.

Huangqi-Danshen Decoction Against Renal Fibrosis in UUO Mice via TGF-ß1 Induced Downstream Signaling Pathway.

Background: Huangqi-Danshen decoction (HDD) is a Chinese medicinal herb pair with good efficacy in treating chronic kidney disease, but its mechanism needs to be clarified. Aim: To uncover the underlying mechanism of HDD antagonizing renal fibrosis through network pharmacology (NP) analysis and experimental validation. Materials and Methods: The chemical components of water extract of HDD were analyzed by combining the ultra-high performance liquid chromatography coupled with Q-Exactive mass spectrum analysis (UHPLC-QE-MS) and HERB database. NP was used to identify core common targets of HDD components and renal fibrosis. Subsequently, male C57BL/6 mice were divided into Sham, unilateral ureteral obstruction (UUO) and UUO+HDD groups. Renal function, histopathology, Western blotting, and immunohistochemistry analyses were used to evaluate the protective effect of HDD on UUO mice. The effects of HDD on signaling pathways were validated in both UUO mice and transforming growth factor-ß1 (TGF-ß1)-induced HK-2 cells. Results: By combining UHPLC-QE-MS analysis and HERB database, 25 components were screened in HDD extract. There were 270 intersection targets of the 25 components and renal fibrosis. Based on their scores in protein-protein interaction analysis and degree values in component-pathway-target triadic network, 6 core common targets of the 25 components and renal fibrosis were identified, namely phosphoinositide 3-kinase (PI3K), signal transducer and activator of transcription 3 (Stat3), non-receptor tyrosine kinase Src (Src), epidermal growth factor receptor (EGFR), matrix metalloproteinase 9 (MMP9), and MMP2. HDD ameliorated renal tubular damage and collagen deposition and downregulated fibrosis-related proteins expression in UUO mice. Furthermore, HDD was demonstrated to reduce PI3K, Stat3, Src, EGFR, and MMP2 expressions, and enhance MMP9 expression in the kidney of UUO mice and in TGF-ß1-induced HK-2 cells. Conclusion: HDD can alleviate renal fibrosis which may be related to regulating the expression of essential proteins in the epithelial-mesenchymal transition and extracellular matrix production/degradation signaling pathways.

The novel peptide PEP-Z-2 potentially treats renal fibrosis in vivo and in vitro by regulating TGF-ß/Smad/AKT/MAPK signaling.

Renal fibrosis is a process in which excessive deposition of extracellular matrix leads to an increase in tissue hardness and gradual destruction of the renal parenchyma. Chronic kidney disease (CKD) commonly progresses to end-stage renal disease (ESRD), ultimately leading to renal failure. This disease has high incidence and mortality rates, but to date, effective treatment options are lacking. PEP-Z-2 is a collagen peptide isolated from redlip croaker scales and may have potential fibroprotective activity. In this study, PEP-Z-2 was found to alleviate unilateral ureteral obstruction (UUO)- and folic acid (FA)-induced kidney injury in a mouse model, reduce collagen deposition in tissues, normalize renal function, reduce the expression of fibrosis markers, reduce reactive oxygen species (ROS) production, and restore the balance of the oxidant/antioxidant system. In vitro experiments also demonstrated that PEP-Z-2 inhibits the TGF-ß-induced differentiation of fibroblasts and renal tubular epithelial cells into myofibroblasts and reduces the production of extracellular matrix (ECM) proteins such as fibronectin, Col I, and α-SMA, demonstrating notable therapeutic effects on renal fibrosis. This effect is achieved by regulating the TGF-ß/Smad/AKT/MAPK pathway. Our research suggested that PEP-Z-2 is a potential therapeutic drug for renal fibrosis, and peptides from aquatic organisms may constitute a new class of candidate drugs for the treatment of renal fibrosis and even other types of organ fibrosis.

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

Bombesin receptor-activated protein (BRAP), encoded by the C6orf89 gene in humans, is expressed in various cells with undefined functions. BC004004, the mouse homologue of C6orf89, has been shown to play a role in bleomycin-induced pulmonary fibrosis through the use of a BC004004 gene knockout mouse (BC004004-/-). In this study, we investigated the potential involvement of BRAP in renal fibrosis using two mouse models: unilateral ureteral obstruction (UUO) and type 2 diabetes mellitus induced by combination of a high-fat diet (HFD) and streptozocin (STZ). BRAP or its homologue was expressed in tubular epithelial cells (TECs) in the kidneys of patients with chronic kidney disease (CKD) and in BC004004+/+ mice. Compared to control mice, BC004004-/- mice exhibited attenuated renal injury and renal fibrosis after UUO or after HFD/STZ treatment. Immunohistochemistry and immunoblot analyses of the kidneys of BC004004+/+ mice after UUO surgery showed a more significant decrease in E-cadherin expression and a more significant increase in both α smooth muscle actin (α-SMA) and vimentin expression compared to BC004004-/- mice. Additionally, stimulation with transforming growth factor-ß1 (TGF-ß1) led to a more significant decrease in E-cadherin expression and a more significant increase in α-SMA and vimentin expression in isolated TECs from BC004004+/+ than in those from BC004004-/- mice. These results suggest that an enhanced epithelial-mesenchymal transition (EMT) process occurred in TECs in BC004004+/+ mice during renal injury, which might contribute to renal fibrosis. The loss of the BRAP homologue in BC004004-/- mice suppressed EMT activation in kidneys and contributed to the suppression of fibrosis during renal injury.

Mechanistic study on lncRNA XIST/miR-124-3p/ITGB1 axis in renal fibrosis in obstructive nephropathy.

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-ß1 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-ß1 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-ß1-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-ß1-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.

Inhibitory effects of Eplerenone on angiogenesis via modulating SGK1/TGF-ß pathway in contralateral kidney of CKD pregnancy rats.

BACKGROUND: Eplerenone is a selective aldosterone receptor blocker that is effective in preventing the progression of chroinic kidney disease (CKD). However, its mechanism and role in CKD pregnancy still remain uncertain. The aim of this study was to evaluate whether eplerenone could attenuated the fibrosis of unilateral ureteral obstruction (UUO) pregnant rats' contralateral kidney, improved pregnancy outcome and explore its therapeutic mechanisms. METHODS: A pregnancy rat model of UUO established, female Wistar rats were randomly assigned into sham-operated group (Sham group),sham-operated combined pregnancy group (SP group), unilateral ureteral obstruction combined pregnancy group (UUO + Pregnancy group), unilateral ureteral obstruction combined pregnancy, administered eplerenone (UUO + Pregnancy+Eplerenone group). On the 18th day of pregnancy, the rats were placed in a metabolic cage, 24 h urine was collected and stored at -80 °C. Next day, all animals were euthanized, and serum was collected by centrifugation and stored at -20 °C. Then the right kidney was extracted, a part of the kidney was placed in 4% paraformaldehyde for morphology, immunohistochemical staining, and immunofluorescence staining, and the other part was placed in a - 80 °C refrigerator for RNA and protein extraction. In vitro, HUVECs was treated with aldosterone, progesterone and estradiol, VEGFA and its receptor blocker bevacizumab. The ability of proliferation, migration and tubularization of HUVECs was detected by CCK-8, scratch wound assay and endothelial tube formation assay. And the co-expression of CD34 and α-SMA of HUVECs was detected by Flow cytometry. RESULTS: Immunofluorescence results showed that the co-expression of CD34 and α-SMA increased in the UUO + Pregnancy group was significantly increased. The expression of SGK-1, TGFß-1, Smad2, Smad3, VEGF-A, VEGFR2, CD34, α-SMA and Collagen I was significantly higher in the kidneys of the UUO + Pregnancy group compared to the Sham group and SP group. Eplerenone inhibited the expression of those results. In vitro, the ability of proliferation, migration and tubularization was increased after treated with aldosterone, aldosterone with progesterone and estradiol or VEGFA. Similarly, the expression of α-SMA on the surface of HUVECs treated with aldosterone, aldosterone with progesterone and estradiol were increased, while eplerenone supressed its expression. CONCLUSION: Eplerenone inhibits renal angiogenesis by blocking the SGK-1/TGFß signal transduction pathway, thereby inhibiting the phenotypic transformation of endothelial cells, slowing down renal fibrosis, and reducing kidney damage caused by pregnancy.

miR-542-5p targets GREM1 to affect the progression of renal fibrosis.

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-ß1 (TGF-ß1)-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-ß1; 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.

Effect of Tissue-derived Angiotensinogen on Kidney Injury and Fibrosis in Obstructive Nephropathy.

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.

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

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.

MiR-92a-3p Knockdown Attenuates Transforming Growth Factor-ß1-induced Tubulointerstitial Fibrosis by Targeting LIN28A-mediated EMT Pathway.

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)-ß1, 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-ß1 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-ß1-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.

CD5L up-regulates the TGF-ß signaling pathway and promotes renal fibrosis.

Renal fibrosis is the common final pathway of progressive renal diseases, in which the macrophages play an important role. ELISA was used to detect CD5 antigen-like (CD5L) in serum samples from end-stage renal disease (ESRD), as well as in mice serum with unilateral ureteral occlusion (UUO). Recombinant CD5L was injected into UUO mice to assess renal injury, fibrosis, and macrophage infiltration. The expression of CD5L was significantly upregulated in the serum of patients with ESRD and UUO mice. Histological analysis showed that rCD5L-treated UUO mice had more severe renal injury and fibrosis. Furthermore, rCD5L promoted the phenotypic transfer of monocytes from Ly6Chigh to LyC6low. RCD5L promoted TGF-ß signaling pathway activation by promoting Smad2/3 phosphorylation. We used Co-IP to identify HSPA5 interact with CD5L on cell membrane could inhibit the formation of the Cripto/HSPA5 complex, and promote the activation of the TGF-ß signaling pathway. The CD5L antibody could reduce the degree of renal fibrosis in UUO mice.

Lipidomic study of kidney in a mouse model with urine flow obstruction.

Obstructed urine flow is known to cause structural and functional kidney damage leading to renal fibrosis. However, limited information is available on the change in kidney lipids during urinary tract obstruction. In this study, we investigated the change in lipidome in a mouse model with unilateral ureteral obstruction (UUO). The establishment of the UUO model was confirmed by histopathological examination using transmission electron microscopy. Untargeted liquid chromatography/mass spectrometry was carried out over a time course of 4 and 7 days. Compared to the sham control, the UUO kidney at 7 days showed dilatation of the renal tubule with loss of brush borders and thickening of the capillary endothelium. In the kidney lipidomes obtained from the UUO 7 days group compared to the control, a significant decrease of ceramide, sphingomyelin, phosphatidylcholine, lysophospholipids, and phosphatidylethanolamine was observed, whereas cholesteryl esters, free fatty acids, phosphatidylglycerol, and cardiolipins were significantly increased. The present study revealed the disturbed lipid metabolism in the UUO model, which may provide a clue to potential lipid pathways and therapeutic targets for the early stage of renal fibrosis.

SFN promotes renal fibrosis via binding with MYH9 in chronic kidney disease.

Chronic kidney disease (CKD) is a clinical syndrome characterized by persistent renal function decline. Renal fibrosis is the main pathological process in CKD, but an effective treatment does not exist. Stratifin (SFN) is a highly-conserved, multi-function soluble acidic protein. Therefore, this study explored the effects of SFN on renal fibrosis. First, we found that SFN was highly expressed in patients with CKD, as well as in renal fibrosis animal and cell models. Next, transforming growth factor-beta 1 (TGF-ß1) induced injury and fibrosis in human renal tubule epithelial cells, and SFN knockdown reversed these effects. Furthermore, SFN knockdown mitigated unilateral ureteral obstruction (UUO)-induced renal tubular dilatation and renal interstitial fibrosis in mice. Liquid chromatography-tandem mass spectrometry/mass spectrometry (LC-MS/MS), co-immunoprecipitation (Co-IP), and immunofluorescence co-localization assays demonstrated that SFN bound the non-muscle myosin-encoding gene, myosin heavy chain 9 (MYH9), in the cytoplasm of renal tubular epithelial cells. MYH9 knockdown also reduced Col-1 and α-SMA expression, which are fibrosis markers. Finally, silencing SFN decreased MYH9 expression, alleviating renal fibrosis. These results suggest that SFN promotes renal fibrosis in CKD by interacting with MYH9. This study may provide potential strategies for the treatment of CKD.

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.

Shenqi Pill Mitigates Renal Interstitial Fibrosis Through Weakening Notch1/Jag1 Pathway.

INTRODUCTION: Shenqi pill (SQP) can be used to treat various kidney related diseases, but its exact mechanism of action remains unclear. We intended to analyze the role and mechanism of SQP on renal interstitial fibrosis (RIF). METHODS: After performing unilateral ureteral obstruction (UUO) surgery following the Institutional Animal Care and Use Committee guidelines, all rats were assigned into the sham group, UUO group, UUO + SQP 1.5 g/kg, UUO + SQP 3 g/kg, and UUO + SQP 6 g/kg groups. After treatment with SQP for 4 weeks, the appearance of kidney, serum creatinine (SCr), and blood urea nitrogen (BUN) levels were monitored in each group. The pathological injury, extracellular matrix (ECM), and Notch1 pathway-related protein levels were measured using H&E staining, Masson staining, immunohistochemistry, and Western blot, respectively. RESULTS: SQP could obviously ameliorate the appearance of the kidney as well as the levels of SCr and BUN in UUO rats (SCr: 67.6 ± 4.64 µM, 59.66 ± 4.96 µM, 48.76 ± 4.44 µM, 40.43 ± 3.02 µM for UUO, low, medium, and high SQP treatment groups; BUN: 9.09 ± 0.97 mM, 7.72 ± 0.61 mM, 5.42 ± 0.42 mM, 4.24 ± 0.34 mM for UUO, low, medium, and high SQP treatment groups; P < .05). SQP also effectively mitigated renal tissue injury in UUO rats (P < .05). Moreover, we uncovered that SQP significantly inhibited Collagen I, α-SMA, Collagen IV, TGF-B1, Notch1, and Jag1 protein expressions in UUO rats kidney (P < .05). CONCLUSION: Our data elucidated that SQP can alleviate RIF, and the mechanism may be related to the Notch1/Jag1 pathway. DOI: 10.52547/ijkd.7703.

Irisin ameliorates UUO-induced renal interstitial fibrosis through TGF-ß1/periostin/MMP-2 signaling pathway.

Renal fibrosis is the most common pathway in progressive kidney diseases. The unilateral ureteral obstruction (UUO) model is used to induce progressive renal fibrosis. We evaluated the effects of irisin on renal interstitial fibrosis in UUO mice. The GSE121190, GSE36496, GSE42303, and GSE96101 datasets were downloaded from the Gene Expression Omnibus (GEO) database. In total, 656 differentially expressed genes (DEGs) were identified in normal and UUO mouse renal samples. Periostin and matrix metalloproteinase-2 (MMP-2) were selected to evaluate the effect of irisin on renal fibrosis in UUO mice. In UUO mice, irisin ameliorated renal function, decreased the expression of periostin and MMP-2, and attenuated epithelial-mesenchymal transition and extracellular matrix deposition in renal tissues. In HK-2 cells, irisin treatment markedly attenuated TGF-ß1-induced expression of periostin and MMP-2. Irisin treatment also inhibited TGF-ß1-induced epithelial-mesenchymal transition, extracellular matrix formation, and inflammatory responses. These protective effects of irisin were abolished by the overexpression of periostin and MMP-2. In summary, irisin treatment can improve UUO-induced renal interstitial fibrosis through the TGF-ß1/periostin/MMP-2 signaling pathway, suggesting that irisin may be used for the treatment of renal interstitial fibrosis.

TREM2 deficiency aggravates renal injury by promoting macrophage apoptosis and polarization via the JAK-STAT pathway in mice.

The triggering receptor expressed on myeloid cells 2 (TREM2) is an immune receptor that affects cellular phenotypes by modulating phagocytosis and metabolism, promoting cell survival, and counteracting inflammation. Its role in renal injury, in particular, unilateral ureteral obstruction (UUO) or ischemia-reperfusion injury (IRI)-induced renal injury remains unclear. In our study, WT and Trem2-/- mice were employed to evaluate the role of TREM2 in renal macrophage infiltration and tissue injury after UUO. Bone marrow-derived macrophages (BMDM) from both mouse genotypes were cultured and polarized for in vitro experiments. Next, the effects of TREM2 on renal injury and macrophage polarization in IRI mice were also explored. We found that TREM2 expression was upregulated in the obstructed kidneys. TREM2 deficiency exacerbated renal inflammation and fibrosis 3 and 7 days after UUO, in association with reduced macrophage infiltration. Trem2-/- BMDM exhibited increased apoptosis and poorer survival compared with WT BMDM. Meanwhile, TREM2 deficiency augmented M1 and M2 polarization after UUO. Consistent with the in vivo observations, TREM2 deficiency led to increased polarization of BMDM towards the M1 proinflammatory phenotype. Mechanistically, TREM2 deficiency promoted M1 and M2 polarization via the JAK-STAT pathway in the presence of TGF-ß1, thereby affecting cell survival by regulating mTOR signaling. Furthermore, cyclocreatine supplementation alleviated cell death caused by TREM2 deficiency. Additionally, we found that TREM2 deficiency promoted renal injury, fibrosis, and macrophage polarization in IRI mice. The current data suggest that TREM2 deficiency aggravates renal injury by promoting macrophage apoptosis and polarization via the JAK-STAT pathway. These findings have implications for the role of TREM2 in the regulation of renal injury that justify further evaluation.

Emodin improves renal fibrosis in chronic kidney disease by regulating mitochondrial homeostasis through the mediation of peroxisome proliferator-activated receptor-gamma coactivator-1 alpha (PGC-1α).

Chronic kidney disease (CKD) is a leading public health issue associated with high morbidity worldwide. However, there are only a few effective therapeutic strategies for CKD. Emodin, an anthraquinone compound from rhubarb, can inhibit fibrosis in tissues and cells. Our study aims to investigate the antifibrotic effect of emodin and the underlying molecular mechanism. A unilateral ureteral obstruction (UUO)-induced rat model was established to evaluate the effect of emodin on renal fibrosis development. Hematoxylin and eosin staining, Masson's trichrome staining, and immunohistochemistry staining were performed to analyze histopathological changes and fibrotic features after emodin treatment. Subsequently, a transforming growth factor-beta 1 (TGF-ß1)-induced cell model was used to assess the inhibition of emodin on cell fibrosis in vitro. Furthermore, Western blot analysis and real-time quantitative reverse transcription-polymerase chain reaction were performed to validate the regulatory mechanism of emodin on renal fibrosis progression. As a result, emodin significantly improved histopathological abnormalities in rats with UUO. The expression of fibrosis biomarkers and mitochondrial biogenesis-related proteins also decreased after emodin treatment. Moreover, emodin blocked TGF-ß1-induced fibrotic phenotype, lipid accumulation, and mitochondrial homeostasis in NRK-52E cells. Conversely, peroxisome proliferator-activated receptor-gamma coactivator-1 alpha (PGC-1α) silencing significantly reversed these features in emodin-treated cells. Collectively, emodin plays an important role in regulating PGC-1α-mediated mitochondria function and energy homeostasis. This indicates that emodin exhibits great inhibition against renal fibrosis and acts as a promising inhibitor of CKD.

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.

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

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