Kidneys Of Unilateral Ureteral Obstruction Mice Research Articles

Huangqi-Danshen Decoction Against Renal Fibrosis in UUO Mice via TGF-β1 Induced Downstream Signaling Pathway.

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. To uncover the underlying mechanism of HDD antagonizing renal fibrosis through network pharmacology (NP) analysis and experimental validation. 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-β1 (TGF-β1)-induced HK-2 cells. 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-β1-induced HK-2 cells. 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.

Astragalus mongholicus bunge and panax notoginseng formula (A&P) improves renal fibrosis in UUO mice via inhibiting the long non-coding RNA A330074K22Rik and downregulating ferroptosis signaling

BackgroundChronic kidney disease (CKD) and its associated end-stage renal disease (ESRD) are significant health problems that pose a threat to human well-being. Renal fibrosis is a common feature and ultimate pathological outcome of various CKD leading to ESRD. The Astragalus mongholicus Bunge and Panax notoginseng formula (A&P) is a refined compound formulated by our research group, which has been clinically administered for over a decade and has demonstrated the ability to improve the inflammatory state of various acute or chronic kidney diseases. However, the underlying mechanism by which A&P ameliorates renal fibrosis remains unclear.MethodsWe established a mouse model by surgically ligating the unilateral ureter to induce renal injury in vivo. And we utilized renal in situ electroporation of a plasmid with low LncRNA A33 expression to establish the unilateral ureteral obstruction(UUO)mouse model. In vitro, we stimulated primary tubular epithelial cells(pTEC) injury using TGF-β1, siRNA-A33, and pcDNA3.1-A33 plasmids were transfected into pTECs to respectively knockdown and overexpress LncRNA A33, and both in vitro and in vivo models were intervened with A&P.ResultsThe results demonstrated that A&P effectively alleviated renal fibrosis in mice. Subsequent findings indicated high expression of LncRNA A33 in the kidneys of UUO mice and TGF-β1-induced renal tubular cells. In situ, renal electroporation of a plasmid with reduced LncRNA A33 expression revealed that inhibiting LncRNA A33 significantly improved renal fibrosis in UUO mice. Moreover, A&P effectively suppressed LncRNA A33 expression both in vitro and in vivo. Subsequent downregulation of LncRNA A33 in renal tubular epithelial cells resulted in the downregulation of numerous fibrotic markers, a significant inhibition of LncRNA A33, and a notable reduction in downstream ferroptosis signaling. Cell experiments demonstrated that A&P improved renal fibrosis in UUO mice by inhibiting LncRNA A33 and downregulating ferroptosis signaling.ConclusionThrough the inhibition of LncRNA A33 and subsequent downregulation of ferroptosis signaling, A&P showed potential as a therapeutic approach for improving renal fibrosis in UUO mice, providing a potential treatment avenue for CKD.

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-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.

Noscapine alleviates unilateral ureteral obstruction-induced inflammation and fibrosis by regulating the TGFβ1/Smads signaling pathways

Renal fibrosis is a common pathway leading to progressive renal function loss in various forms of chronic kidney disease. Many fibrogenic factors regulate renal fibrosis; two key players are post-injury inflammation and transforming growth factor-β1 (TGF-β1)-induced myofibroblast differentiation. Myofibroblast differentiation is tightly regulated by the microtubule polymerization. Noscapine, an antitussive plant alkaloid, is a potent microtubule-interfering agent previously identified as a potential anticancer compound. Here, we examined how noscapine affects renal fibrogenesis in an in vitro renal fibroblast model and an in vivo unilateral ureteral obstruction (UUO) model. UUO mice were intraperitoneally treated with noscapine at 1 day before UUO surgery and daily thereafter. At 7 days post-surgery, kidneys were collected for further analysis. To analyze whether noscapine inhibits downstream TGF-β1-related signaling, we pre-incubated NRK-49F fibroblasts with noscapine and then performed TGF-β1 stimulation. In UUO mice, noscapine attenuated extracellular matrix protein deposition and the expression levels of type I collagen, type IV collagen, α-smooth muscle actin, and fibronectin. In addition, noscapine decreased tubulointerstitial inflammation in UUO kidneys by reducing TLR2 expression, modulating NLRP3 inflammasome activation, reducing macrophage infiltration, and antagonizing the M2 macrophage phenotype. Furthermore, noscapine pre-incubation suppressed the TGF-β1-induced fibroblast-myofibroblast transformation by downregulating the TGF-β/Smads signaling pathways in NRK-49F cells. These results suggest that noscapine reduces tubulointerstitial inflammation and fibrosis in the kidneys of UUO mice and inhibits the fibroblast-myofibroblast transformation induced by TGF-β1. Noscapine is an over-the-counter antitussive that has been used safely for several decades. Therefore, noscapine is an attractive therapeutic agent for inhibiting renal tubulointerstitial fibrosis.

Alpha-cyano-4-hydroxycinnamic acid alleviates renal fibrosis and inflammation in chronic kidney disease

Renal fibrosis is a crucial pathological phenomenon of chronic kidney disease (CKD) that contributes to the progressive loss of renal function. Monocarboxylate transporter 1 (MCT1) plays an important role in clear cell renal cell carcinoma; however, the role of the MCT1 inhibitor alpha-cyano-4-hydroxycinnamic acid (α-CHCA) in renal fibrosis remains unclear. In this study, we evaluated the role of α-CHCA, an MCT1 inhibitor, in treating renal fibrosis associated with CKD. We used α-CHCA in a CKD mouse model of unilateral ureteral obstruction (UUO), and mouse proximal tubular epithelial cells (mPTCs) stimulated by transforming growth factor-β1 (TGF-β1). In vivo, administration of α-CHCA improved tubulointerstitial fibrosis, which was consistent with the reduced expression of collagen I/III, fibronectin (FN1), and α-smooth muscle actin (α-SMA) in the kidneys of UUO mice. Similarly, the overexpression of inflammatory factors in UUO kidneys decreased in response to α-CHCA treatment. In vitro, α-CHCA pretreatment inhibited the induction of collagen I/III, FN1, and α-SMA production in mPTCs treated with TGF-β1. Collectively, these findings indicate that α-CHCA may play a therapeutic role in renal fibrosis associated with CKD by inhibiting collagen deposition and subsequent fibrosis and inflammation. Highlights Chronic kidney disease (CKD) mice have increased MCT1 expression. α-CHCA, a MCT1 inhibitor, alleviates renal fibrosis in CKD mice. α-CHCA improves the inflammation in CKD mice. α-CHCA inhibits TGF-β1-induced extracellular matrix secretion of proximal tubular epithelial cells.

Renoprotective Effect of TP0472993, a Novel and Selective 20-Hydroxyeicosatetraenoic Acid Synthesis Inhibitor, in Mouse Models of Renal Fibrosis.

Kidney fibrosis is considered the essential pathophysiological process for the progression of chronic kidney disease (CKD) toward renal failure. 20-Hydroxyeicosatetraenoic acid (20-HETE) has crucial roles in modulating the vascular response in the kidney and the progression of albuminuria. However, the roles of 20-HETE in kidney fibrosis are largely unexplored. In the current research, we hypothesized that if 20-HETE has important roles in the progression of kidney fibrosis, 20-HETE synthesis inhibitors might be effective against kidney fibrosis. To verify our hypothesis, this study investigated the effect of a novel and selective 20-HETE synthesis inhibitor, TP0472993, on the development of kidney fibrosis after folic acid- and obstructive-induced nephropathy in mice. Chronic treatment with TP0472993 at doses of 0.3 and 3 mg/kg twice a day attenuated the degree of kidney fibrosis in the folic acid nephropathy and the unilateral ureteral obstruction (UUO) mice, as demonstrated by reductions in Masson's trichrome staining and the renal collagen content. In addition, TP0472993 reduced renal inflammation, as demonstrated by markedly reducing interleukin-1β (IL-1β) and tumor necrosis factor alpha (TNF-α) levels in the renal tissue. Chronic treatment with TP0472993 also reduced the activity of extracellular signal-regulated kinase 1/2 (ERK1/2) and signal transducer and activator of transcription 3 (STAT3) in the kidney of UUO mice. Our observations indicate that inhibition of 20-HETE production with TP0472993 suppresses the kidney fibrosis progression via a reduction in the ERK1/2 and STAT3 signaling pathway, suggesting that 20-HETE synthesis inhibitors might be a novel treatment option against CKD. SIGNIFICANCE STATEMENT: In this study, we demonstrate that the pharmacological blockade of 20-hydroxyeicosatetraenoic acid (20-HETE) synthesis using TP0472993 suppresses the progression of kidney fibrosis after folic acid- and obstructive-induced nephropathy in mice, indicating that 20-HETE might have key roles in the pathogenesis of kidney fibrosis. TP0472993 has the potential to be a novel therapeutic approach against chronic kidney disease.

Pemafibrate inhibited renal dysfunction and fibrosis in a mouse model of adenine-induced chronic kidney disease

AimsPeroxisome proliferator-activated receptor-alpha (PPARα) levels are markedly lower in the kidneys of chronic kidney disease (CKD) patients. Fibrates (PPARα agonists) are therapeutic agents against hypertriglyceridemia and potentially against CKD. However, conventional fibrates are eliminated by renal excretion, limiting their use in patients with impaired renal function. Here, we aimed to evaluate the renal risks associated with conventional fibrates via clinical database analysis and investigate the renoprotective effects of pemafibrate, a novel selective PPARα modulator mainly excreted into the bile. Main methodsThe risks associated with conventional fibrates (fenofibrate, bezafibrate) to the kidneys were evaluated using the Food and Drug Administration Adverse Event Reporting System. Pemafibrate (1 or 0.3 mg/kg/day) was administered daily using an oral sonde. Its renoprotective effects were examined in unilateral ureteral obstruction (UUO)-induced renal fibrosis model mice (UUO mice) and adenine-induced CKD model mice (CKD mice). Key findingsThe ratios of glomerular filtration rate decreased and blood creatinine increased were markedly higher after conventional fibrate use. Pemafibrate administration suppressed increased gene expressions of collagen-I, fibronectin, and interleukin 1 beta (IL-1β) in the kidneys of UUO mice. In CKD mice, it suppressed increased plasma creatinine and blood urea nitrogen levels and decreased red blood cell count, hemoglobin, and hematocrit levels, along with renal fibrosis. Moreover, it inhibited the upregulation of monocyte chemoattractant protein-1, IL-1β, tumor necrosis factor-alpha, and IL-6 in the kidneys of CKD mice. SignificanceThese results demonstrated the renoprotective effects of pemafibrate in CKD mice, confirming its potential as a therapeutic agent for renal disorders.

Antioxidant, Anti-Apoptotic, and Anti-Inflammatory Effects of Farrerol in a Mouse Model of Obstructive Uropathy.

Obstructive uropathy is a clinical condition that can lead to chronic kidney disease. However, treatments that can prevent the progression of renal injury and fibrosis are limited. Farrerol (FA) is a natural flavone with potent antioxidant and anti-inflammatory properties. Here, we investigated the effect of FA on renal injury and fibrosis in a mouse model of unilateral ureteral obstruction (UUO). Mice underwent a sham or UUO operation and received intraperitoneal injections of FA (20 mg/kg) daily for 8 consecutive days. Histochemistry, immunohistochemistry and immunofluorescence staining, TdT-mediated dUTP nick end labeling assay, Western blotting, gene expression analysis, and biochemical tests were performed. FA attenuated renal dysfunction (p < 0.05) and ameliorated renal tubular injury (p < 0.01) and interstitial fibrosis (p < 0.001) in UUO mice. FA alleviated 4-hydroxynonenal expression (p < 0.001) and malondialdehyde levels (p < 0.01) by regulating pro-oxidant and antioxidant enzymes. Apoptosis in the kidneys of UUO mice was inhibited by FA (p < 0.001), and this action was accompanied by decreased expression of cleaved caspase-3 (p < 0.01). Moreover, FA alleviated pro-inflammatory cytokine production (p < 0.001) and macrophage infiltration (p < 0.01) in the kidneys of UUO mice. These results suggest that FA ameliorates renal injury and fibrosis in the UUO model by inhibiting oxidative stress, apoptosis, and inflammation.

MiR−122−5p Regulates Renal Fibrosis In Vivo

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.

Protective Effects of Carnosol on Renal Interstitial Fibrosis in a Murine Model of Unilateral Ureteral Obstruction.

Renal fibrosis is a common feature of chronic kidney disease and is a promising therapeutic target. However, there is still limited treatment for renal fibrosis, so the development of new anti-fibrotic agents is urgently needed. Accumulating evidence suggest that oxidative stress and endoplasmic reticulum (ER) stress play a critical role in renal fibrosis. Carnosol (CS) is a bioactive diterpene compound present in rosemary plants and has potent antioxidant and anti-inflammatory properties. In this study, we investigated the potential effects of CS on renal injury and fibrosis in a murine model of unilateral ureteral obstruction (UUO). Male C57BL/6J mice underwent sham or UUO surgery and received intraperitoneal injections of CS (50 mg/kg) daily for 8 consecutive days. CS improved renal function and ameliorated renal tubular injury and interstitial fibrosis in UUO mice. It suppressed oxidative injury by inhibiting pro-oxidant enzymes and activating antioxidant enzymes. Activation of ER stress was also attenuated by CS. In addition, CS inhibited apoptotic and necroptotic cell death in kidneys of UUO mice. Furthermore, cytokine production and immune cell infiltration were alleviated by CS. Taken together, these findings indicate that CS can attenuate renal injury and fibrosis in the UUO model.

Investigation into the effect and mechanism of dapagliflozin against renal interstitial fibrosis based on transcriptome and network pharmacology

BackgroundRenal interstitial fibrosis (RIF) is the final pathway for chronic kidney diseases (CKD) to end-stage renal disease (ESRD). Dapagliflozin, a selective inhibitor of the sodium glucose co-transporter 2, reduced the risk of renal events in non-diabetic CKD patients in the DAPA-CKD trial. However, the effect and mechanism of dapagliflozin on RIF are not very clear. Currently, we evaluate the effects of dapagliflozin on RIF and systematically explore its mechanism. Methods and ResultsFirstly, unilateral ureteral obstruction (UUO) mouse model was established to evaluate effects of dapagliflozin on RIF, and results demonstrated dapagliflozin improved renal function and RIF of UUO mice independent of blood glucose control. Subsequently, transcriptome analysis was performed to explore the potential mechanism of dapagliflozin against RIF, which exhibited the therapeutic effect of dapagliflozin on RIF may be achieved through multiple pathways regulation. Then we verified the potential mechanisms with molecular biology methods, and found that dapagliflozin treatment significantly alleviated inflammation, apoptosis, oxidative stress and mitochondrial injury in kidneys of UUO mice. Furthermore, network pharmacology analysis was used to investigate the potential targets of dapagliflozin against RIF. Moreover, we also applied molecular docking and molecular dynamics simulation to predict the specific binding sites and binding capacity of dapagliflozin and hub target. ConclusionsDapagliflozin had therapeutic effect on RIF independent of blood glucose control, and the protective effects probably mediated by multiple pathways and targets regulation.

The Polysaccharide of Ligustrum Lucidum Ait. Protected from Renal Fibrosis by Interfering with Angiotensin II Signaling

BackgroundA number of preclinical advances have indicated the cellular and molecular mechanisms underlying renal fibrosis, such as the signaling of angiotensin II (Ang II), which is a bioactive peptide in renin‐angiotensin system. As one of kidney‐tonifying herbs, Fructus Ligustri Lucidi (FLL), the fruit of Ligustrum lucidum Ait. (Oleaceae) recorded as a tonic and dietary supplement, is a commonly prescribed component in traditional kidney‐tonifying herbal formula. This study aimed to clarify the polysaccharide component in FLL and investigate its potential role in preservation against renal fibrosis.MethodsThe crude polysaccharide of FLL was prepared and deproteinized by Sevag reagent. After lyophilization, the DEAE‐cellulose DE52 column and the Sephacryl S‐200 HR column were used for further isolation and purification of homogenous polysaccharide. The mice with unilateral ureteral obstruction (UUO) underwent daily intraperitoneal injection with FLL polysaccharide starting 7 days before UUO surgery until day 7 after surgery. The murine mesangial SV40‐MES13 cells were treated with vehicle, Ang II (10‐7 M) either with the absence and presence of angiotensin type I receptor blocker (ARB) (10‐6M), or with low dose (50 μg/mL, Poly‐L) and high dose (200 μg/mL, Poly‐H) of FLL polysaccharide.ResultsThe purified homogenous polysaccharide (Poly) was explored from FLL and the carbohydrate content was 97.45%. A hypothetical structure for Poly was proposed considering the comprehensive data of methylation, monosaccharides, and NMR analyses. Treatment of UUO mice with Poly decreased kidney index and ameliorated glomerular sclerosis and interstitial fibrosis, as well as down‐regulated the expression of profibrogenic factor VEGF and matrix fibrotic protein fibronectin (FN). Furthermore, as the same effect to ARB, the pre‐incubation with Poly alleviated the Ang II‐induced up‐regulation of FN in mesangial cells by immunofluoresence staining. The protein expressions of FN and proinflammatory factors like MCP‐1 and Rantes were significantly lower in Poly‐treated cell group than those in Ang II treatment group.ConclusionThe polysaccharide of Ligustrum lucidum Ait. was accurately explored with structure clarification. This polysaccharide component repressed angiotensin II‐induced fibrotic phenotype of renal mesangial cells and exerted beneficial effects on kidney of UUO mice.

ZLN005 Alleviates In Vivo and In Vitro Renal Fibrosis via PGC-1α-Mediated Mitochondrial Homeostasis.

Currently, chronic kidney disease (CKD) is one of the most common diseases; it is also a serious threat to human health due to its high mortality, and its treatment is still a major clinical challenge. Mitochondrial dyshomeostasis plays an important role in the development of CKD. ZLN005 is a novel peroxisome-proliferator-activated receptor-γ coactivator-1α (PGC-1α) activator from our laboratory. To explore whether ZLN005 can protect against CKD in vivo and in vitro, a unilateral ureteral obstruction (UUO) model and TGF-β1-treated renal tubular epithelial cells (TECs), respectively, were used in this study. We found that ZLN005-administrated UUO mice showed less kidney damages than control mice, as indicated by the reduced expression of fibrotic biomarkers in the kidney of UUO mice. ZLN005 treatment also alleviated the TGF-β1-induced fibrotic phenotype and lipid accumulation in TECs. Our study demonstrated ZLN005 treatment improved mitochondrial homeostasis at least partially via the activation of PGC-1α, thus maintaining mitochondria function and energy homeostasis. In summary, ZLN005 treatment ameliorates UUO-induced renal fibrosis, providing conceptional support for mitochondria-targeting therapies for chronic kidney disease.

Dihydroartemisinin suppresses renal fibrosis in mice by inhibiting DNA-methyltransferase 1 and increasing Klotho.

Renal fibrosis is an unavoidable end result of all forms of progressive chronic kidney diseases (CKD). Discovery of efficacious drugs against renal fibrosis is in crucial need. In a preliminary study we found that a derivative of artemisinin, dihydroartemisinin (DHA), exerted strong renoprotection, and reversed renal fibrosis in adenine-induced CKD mouse model. In this study we investigated the anti-fibrotic mechanisms of DHA, particularly its specific target in renal cells. Renal fibrosis was induced in mice by unilateral ureteral obstruction (UUO) or oral administration of adenine (80 mg · kg-1), the mice received DHA (30 mg · kg-1 · d-1, i.g.) for 14 or 21 days, respectively. We showed that DHA administration markedly attenuated the inflammation and fibrotic responses in the kidneys and significantly improved the renal function in both the renal fibrosis mouse models. In adenine-treated mice, DHA was more effective than 5-azacytidine against renal fibrosis. The anti-fibrotic effects of DHA were also observed in TGF-β1-treated HK-2 cells. In order to determine the target protein of DHA, we conducted pull-down technology coupled with shotgun proteomics using a small-molecule probe based on the structure of DHA (biotin-DHA). As a results, DNA methyltransferase 1 (DNMT1) was identified as the anti-fibrotic target of DHA in 3 different types of renal cell lines (HK-2, HEK293 and 3T3). We demonstrated that DHA directly bound to Asn 1529 and Thr 1528 of DNMT1 with a Kd value of 8.18 μM. In primary mouse renal tubular cells, we showed that DHA (10 μM) promoted DNMT1 degradation via the ubiquitin-proteasome pathway. DHA-reduced DNMT1 expression effectively reversed Klotho promoter hypermethylation, which led to the reversal of Klotho protein loss in the kidney of UUO mice. This subsequently resulted in inhibition of the Wnt/β-catenin and TGF-β/Smad signaling pathways and consequently conferred renoprotection in the animals. Knockdown of Klotho abolished the renoprotective effect of DHA in UUO mice. Our study reveals a novel pharmacological activity for DHA, i.e., renoprotection. DHA exhibits this effect by targeting DNMT1 to reverse Klotho repression. This study provides an evidence for the possible clinical application of DHA in the treatment of renal fibrosis.

The mmu_circRNA_37492/hsa_circ_0012138 function as potential ceRNA to attenuate obstructive renal fibrosis

Circular RNAs (circRNAs) are involved in the pathogenesis of certain renal diseases, however, the function and mechanism of them in renal fibrosis remains largely unknown. In the present study, RNA expression data in unilateral ureteral obstruction (UUO) kidneys was obtained from our previous circRNA Microarray and public Gene Expression Omnibus datasets to construct a ceRNA network. The effects of target circRNA as long as the homologous human circRNA on renal fibrosis was examined in vitro and in vivo. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was further performed among genes regulated by the human circRNA. We found that circRNA_37492, showing well connection degree in the ceRNA network, was abundant expression and high sequence conservation. We observed that the expression of circRNA_37492 was induced by the TGF-β1 or UUO in BUMPT cells and C57BL/6 mice, respectively. In vitro, cytoplasmic circRNA_37492 inhibited type I, III collagen and fibronectin deposition by sponging miR-7682-3p and then upregulated its downstream target Fgb. In vivo, overexpression of circRNA_37492 attenuated fibrotic lesions in the kidneys of UUO mice via targeting miR-7682-3p/Fgb axis. Furthermore, hsa_circ_0012138, homologous with circRNA_37492, may potentially target miR-651-5p/FGB axis in human renal fibrosis. Not only that, GO and KEGG enrichment revealed that hsa_circ_0012138-regulated genes were previously demonstrated to related to the fibrosis. In conclusion, we for the first time demonstrated that circRNA_37492 attenuated renal fibrosis via targeting miR-7682-3p/Fgb axis, and the homologous hsa_circRNA_0012138 was speculated as a possible ceRNA to regulate multiple gene expressions and involve in human renal fibrosis, suggesting that circRNA_37492/hsa_circ_0012138 may serve as potent therapy target for obstructive renal fibrosis disease.

Discovery of indol-6-yl-pyrrolo[2,3-c]pyridin-7-one derivatives as bromodomain-containing protein 4 (BRD4) inhibitors for the treatment of kidney fibrosis

Uncovering new therapeutics for kidney fibrosis hold promise for chronic kidney disease (CKD). Considerable studies confirmed that BRD4 inhibition ameliorated kidney injury and fibrosis. In the study, we synthesized a series of indol-6-yl-pyrrolo[2,3-c]pyridin-7-one derivatives and biologically evaluated against BRD4 for structure-activity relationship (SAR). Notably, compound 3r (ZLD2218) exhibited the most potent inhibitory activity against BRD4, with the IC50 value of 107 nM, which was comparative to 92 nM of positive control JQ-1. Importantly, at the dose of 15 and 30 mg/kg/d for consecutive 8 days, ZLD2218 alleviated kidney injury and fibrosis in unilateral ureteral obstruction (UUO) mice, with the 30 mg/kg/d being competitive to 100 mg/kd/d of JQ1. Mechanically, ZLD2218 inhibited BRD4 expression and further suppressed fibrotic signaling in the kidneys of UUO mice and TGF-β1-stimulated TCMK-1 cells. Furthermore, ZLD2218 at the dose of 30 mg/kg/d for 8 days to C57BL/6J mice did not affect liver, kidney function and organ pathological changes. Collectively, compound 3r (ZLD2218) might be a promising lead compound of BRD4 inhibitor for the treatment of kidney fibrosis.

Nicotinamide N-methyltransferase ameliorates renal fibrosis by its metabolite 1-methylnicotinamide inhibiting the TGF-β1/Smad3 pathway.

Chronic kidney disease (CKD), a disease involving damage to the kidney structure and function, is a global public health problem. Tubulointerstitial fibrosis (TIF) is both an inevitable pathological change in individuals with CKD and a driving force in the progression of renal fibrosis. Nicotinamide N-methyltransferase (NNMT) and its metabolite 1-methylnicotinamide (MNAM) have been shown to protect against lipotoxicity-induced kidney tubular injury. However, the biological roles of NNMT and MNAM in regulating TIF remain elusive. This study aimed to investigate the protective effect of NNMT and MNAM on TIF and the mechanisms involved. We explored the functions and mechanisms of NNMT and MNAM in TIF, as well as the interaction between NNMT and MNAM, using unilateral ureteral obstruction (UUO) mice and cultured mouse tubular epithelial cells (mTECs) stimulated with transforming growth factor-β1 (TGF-β1). Several important findings were obtained as follows: (1) NNMT expression was upregulated in the kidneys of UUO mice and TGF-β1-induced mTECs, and this upregulation was proposed to be a protective compensatory response to TIF. (2) MNAM was a potentially effective antifibrotic and anti-inflammatory medication in UUO mice. (3) The antifibrotic effect of NNMT overexpression was exerted by increasing the concentration of MNAM. (4) The renoprotective role of MNAM depended on the selective blockade of the interaction of Smad3 with TGFβ receptor I. Overall, our study shows that NNMT is involved in the development and progression of CKD and that its metabolite MNAM may be a novel inhibitor of the TGF-β1/Smad3 pathway with great therapeutic potential for CKD.

Rosmarinic acid ameliorates renal interstitial fibrosis by inhibiting the phosphorylated-AKT mediated epithelial-mesenchymal transition in vitro and in vivo.

Indoxyl sulfate (IS), a uremic toxin, causes chronic kidney disease (CKD) progression via renal fibrosis. Epithelial-mesenchymal transition (EMT) is a crucial feature of renal fibrosis. Rosmarinic acid (RA) is an ester of caffeic acid and 3,4-dihydroxyphenylacetic acid with a wide range of desirable biological activities. In this study, we investigated whether RA exerted anti-renal fibrosis effects and its related mechanisms in a unilateral ureteral obstruction (UUO) mouse model. C57BL/6 mice were orally administered RA (10 and 20 mg kg-1 d-1) for 7 consecutive days before and after UUO surgery. The mice were then sacrificed to collect the blood and kidneys. Hematoxylin and eosin (H&E) and Masson's trichrome staining were used to evaluate the renal injury and function. Immunohistochemical analysis, reverse transcription-polymerase chain reaction (RT-PCR), and western blotting were used to detect the expression levels of EMT markers. In vitro studies were performed using the IS-stimulated NRK-52E cell line. Here, the pathological changes, collagen deposition, and mRNA and protein expression levels of profibrotic factors and fibrotic markers were found to be significantly elevated in the kidneys of UUO mice. We found that RA administration significantly ameliorated UUO-induced kidney damage by reversing abnormal serum creatinine and blood urea nitrogen levels. It was found that RA treatment decreased the expression levels of alpha-smooth muscle actin (α-SMA), collagen I, fibronectin, transforming growth factor (TGF)-β1, vimentin and phosphorylated AKT (p-AKT) while increasing the E-cadherin expression in both UUO kidneys and IS-treated NRK-52E cells. Our results demonstrate that RA may be a promising therapeutic agent for renal interstitial fibrosis.

SAP130 released by damaged tubule drives necroinflammation via miRNA-219c/Mincle signaling in acute kidney injury

Tubules injury and immune cell activation are the common pathogenic mechanisms in acute kidney injury (AKI). However, the exact modes of immune cell activation following tubule damage are not fully understood. Here we uncovered that the release of cytoplasmic spliceosome associated protein 130 (SAP130) from the damaged tubular cells mediated necroinflammation by triggering macrophage activation via miRNA-219c(miR-219c)/Mincle-dependent mechanism in unilateral ureteral obstruction (UUO) and cisplatin-induced AKI mouse models, and in patients with acute tubule necrosis (ATN). In the AKI kidneys, we found that Mincle expression was tightly correlated to the necrotic tubular epithelial cells (TECs) with higher expression of SAP130, a damaged associated molecule pattern (DAMP), suggesting that SAP130 released from damaged tubular cells may trigger macrophage activation and necroinflammation. This was confirmed in vivo in which administration of SAP130-rich supernatant from dead TECs or recombinant SAP130 promoted Mincle expression and macrophage accumulation which became worsen with profound tubulointerstitial inflammation in LPS-primed Mincle WT mice but not in Mincle deficient mice. Further studies identified that Mincle was negatively regulated via miR-219c-3p in macrophages as miR-219c-3p bound Mincle 3′-UTR to inhibit Mincle translation. Besides, lentivirus-mediated renal miR-219c-3p overexpression blunted Mincle and proinflammatory cytokine expression as well as macrophage infiltration in the inflamed kidney of UUO mice. In conclusion, SAP130 is released by damaged tubules which elicit Mincle activation on macrophages and renal necroinflammation via the miR-219c-3p-dependent mechanism. Results from this study suggest that targeting miR-219c-3p/Mincle signaling may represent a novel therapy for AKI.

BMPER Ameliorates Renal Fibrosis by Inhibiting Tubular Dedifferentiation and Fibroblast Activation.

Tubulointerstitial fibrosis is both a pathological manifestation of chronic kidney disease and a driving force for the progression of kidney disease. A previous study has shown that bone morphogenetic protein-binding endothelial cell precursor-derived regulator (BMPER) is involved in lung fibrogenesis. However, the role of BMPER in renal fibrosis remains unknown. In the present study, the expression of BMPER was examined by real-time PCR, Western blot and immunohistochemical staining. The in vitro effects of BMPER on tubular dedifferentiation and fibroblast activation were analyzed in cultured HK-2 and NRK-49F cells. The in vivo effects of BMPER were dissected in unilateral ureteral obstruction (UUO) mice by delivery of BMPER gene via systemic administration of plasmid vector. We reported that the expression of BMPER decreased in the kidneys of UUO mice and HK-2 cells. TGF-β1 increased inhibitor of differentiation-1 (Id-1) and induced epithelial mesenchymal transition in HK-2 cells, and knockdown of BMPER aggravated Id-1 up-regulation, E-cadherin loss, and tubular dedifferentiation. On the contrary, exogenous BMPER inhibited Id-1 up-regulation, prevented E-cadherin loss and tubular dedifferentiation after TGF-β1 exposure. In addition, exogenous BMPER suppressed fibroblast activation by hindering Erk1/2 phosphorylation. Knockdown of low-density lipoprotein receptor-related protein 1 abolished the inhibitory effect of BMPER on Erk1/2 phosphorylation and fibroblast activation. Moreover, delivery of BMPER gene improved renal tubular damage and interstitial fibrosis in UUO mice. Therefore, BMPER inhibits TGF-β1-induced tubular dedifferentiation and fibroblast activation and may hold therapeutic potential for tubulointerstitial fibrosis.