Calycosin inhibited MIF-mediated inflammatory chemotaxis of macrophages to ameliorate ischemia reperfusion-induced acute kidney injury.

BACKGROUND: Inflammatory macrophage infiltration plays a critical role in acute kidney disease induced by ischemia-reperfusion (IRI-AKI). Calycosin is a natural flavone with multiple bioactivities. This study aimed to investigate the therapeutic role of calycosin in IRI-AKI and its underlying mechanism. METHODS: The renoprotective and anti-inflammatory effects of calycosin were analyzed in C57BL/6 mice with IRI-AKI and lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. RNA-seq was used for mechanism investigation. The molecular target of calycosin was screened by in silico methods and validated by surface plasmon resonance (SPR). Macrophage chemotaxis was analyzed using Transwell and agarose gel spot assays. RESULTS: Calycosin treatment significantly reduced serum creatinine and urea nitrogen and attenuated tubular destruction in IRI-AKI mice. Additionally, calycosin markedly suppressed NF-κB signaling activation and the expression of inflammatory mediators IL-1ß and TNF-α in IRI-AKI kidneys and LPS-stimulated RAW 264.7 cells. Interestingly, RNA-seq revealed calycosin remarkably downregulated chemotaxis-related pathways in RAW 264.7 cells. Among the differentially expressed genes, Ccl2/MCP-1, a critical chemokine mediating macrophage inflammatory chemotaxis, was downregulated in both LPS-stimulated RAW 264.7 cells and IRI-AKI kidneys. Consistently, calycosin treatment attenuated macrophage infiltration in the IRI-AKI kidneys. Importantly, in silico target prediction, molecular docking, and SPR assay demonstrated that calycosin directly binds to macrophage migration inhibitory factor (MIF). Functionally, calycosin abrogated MIF-stimulated NF-κB signaling activation and Ccl2 expression and MIF-mediated chemotaxis in RAW 264.7 cells. CONCLUSIONS: In summary, calycosin attenuates IRI-AKI by inhibiting MIF-mediated macrophage inflammatory chemotaxis, suggesting it could be a promising therapeutic agent for the treatment of IRI-AKI.

Research on the global trends of COVID-19 associated acute kidney injury: a bibliometric analysis.

Critically ill COVID-19 patients may exhibit various clinical symptoms of renal dysfunction including severe Acute Kidney Injury (AKI). Currently, there is a lack of bibliometric analyses on COVID-19-related AKI. The aim of this study is to provide an overview of the current research status and hot topics regarding COVID-19 AKI. The literature was retrieved from the Web of Science Core Collection (WoSCC) database. Subsequently, we utilized Microsoft Excel, VOSviewer, Citespace, and Pajek software to revealed the current research status, emerging topics, and developmental trends pertaining to COVID-19 AKI. This study encompassed a total of 1507 studies on COVID-19 AKI. The United States, China, and Italy emerged as the leading three countries in terms of publication numbers, contributing 498 (33.05%), 229 (15.20%), and 140 (9.29%) studies, respectively. The three most active and influential institutions include Huazhong University of Science and Technology, Wuhan University and Harvard Medical School. Ronco C from Italy, holds the record for the highest number of publications, with a total of 15 papers authored. Cheng YC's work from China has garnered the highest number of citations, totaling 470 citations. The co-occurrence analysis of author keywords reveals that 'mortality', 'intensive care units', 'chronic kidney disease', 'nephrology', 'renal transplantation', 'acute respiratory distress syndrome', and 'risk factors' emerge as the primary areas of focus within the realm of COVID-19 AKI. In summary, this study analyzes the research trends in the field of COVID-19 AKI, providing a reference for further exploration and research on COVID-19 AKI mechanisms and treatment.

Screening of active components in Astragalus mongholicus Bunge and Panax notoginseng formula for anti-fibrosis in CKD: nobiletin inhibits Lgals1/PI3K/AKT signaling to improve renal fibrosis.

The Astragalus mongholicus Bunge and Panax notoginseng formula (A&P) has been clinically shown to effectively slow down the progression of chronic kidney disease (CKD) and has demonstrated significant anti-fibrosis effects in experimental CKD model. However, the specific active ingredients and underlying mechanism are still unclear. The active ingredients of A&P were analyzed by Ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-HR-MS). A mouse model of CKD was constructed by 5/6 nephrectomy. Renal function was assessed by creatinine and urea nitrogen. Real-time PCR and Western Blot were performed to detect the mRNA and protein changes in kidney and cells. An in vitro fibrotic cell model was constructed by TGF-ß induction in TCMK-1 cells. The results showed that thirteen active ingredients of A&P were identified by UPLC-HR-MS, nine of which were identified by analysis with standards, among which the relative percentage of NOB was high. We found that NOB treatment significantly improved renal function, pathological damage and reduced the expression level of fibrotic factors in CKD mice. The results also demonstrated that Lgals1 was overexpressed in the interstitial kidney of CKD mice, and NOB treatment significantly reduced its expression level, while inhibiting PI3K and AKT phosphorylation. Interestingly, overexpression of Lgals1 significantly increased fibrosis in TCMK1 cells and upregulated the activity of PI3K and AKT, which were strongly inhibited by NOB treatment. NOB is one of the main active components of A&P. The molecular mechanism by which NOB ameliorates renal fibrosis in CKD may be through the inhibition of Lgals1/PI3K/AKT signaling pathway.

Calycosin pretreatment enhanced the therapeutic efficacy of mesenchymal stem cells to alleviate unilateral ureteral obstruction-induced renal fibrosis by inhibiting necroptosis.

Bone marrow-derived mesenchymal stem cells (MSCs) show antifibrotic activity in various chronic kidney diseases. Here, we aimed to investigate whether Calycosin (CA), a phytoestrogen, could enhance the antifibrotic activity of MSCs in primary tubular epithelial cells (PTECs) induced by TGF-ß1 and in a mouse model of unilateral ureteral obstruction (UUO). We found that MSCs treatment significantly inhibited fibrosis, and CA pretreatment enhanced the effects of MSCs on fibrosis in vitro. Consistent with the in vitro studies, MSCs alleviated tubular injury and renal fibrosis in mice after UUO, and CA-pretreated MSCs resulted in more significant improvements in tubular injury and renal fibrosis than MSCs after UUO. Moreover, MSCs treatment significantly inhibited necroptosis by repressing the elevation of MLKL, RIPK1, and RIPK3 in PTECs treated by TGF-ß1and in mice after UUO, and CA-pretreated MSCs were superior to MSCs in alleviating necroptosis. MSCs significantly reduced TNF-α and TNFR1 expression induced by TGF-ß1 in PTECs and inhibited TGF-ß1, TNF-α, and TNFR1 expression induced by UUO in mice. These effects of MSCs were significantly enhanced after CA pretreatment. Therefore, our results suggest that CA pretreatment enhances the antifibrotic activity of MSCs by inhibiting TGF-ß1/TNF-α/TNFR1 signaling-induced necroptosis.

Artesunate relieves acute kidney injury through inhibiting macrophagic Mincle-mediated necroptosis and inflammation to tubular epithelial cell.

Artesunate is a widely used derivative of artemisinin for malaria. Recent researches have shown that artesunate has a significant anti-inflammatory effect on many diseases. However, its effect on acute kidney injury with a significant inflammatory response is not clear. In this study, we established a cisplatin-induced AKI mouse model and a co-culture system of BMDM and tubular epithelial cells (mTEC) to verify the renoprotective and anti-inflammatory effects of artesunate on AKI, and explored the underlying mechanism. We found that artesunate strongly down-regulated the serum creatinine and BUN levels in AKI mice, reduced the necroptosis of tubular cells and down-regulated the expression of the tubular injury molecule Tim-1. On the other hand, artesunate strongly inhibited the mRNA expression of inflammatory cytokines (IL-1ß, IL-6 and TNF-α), protein levels of inflammatory signals (iNOS and NF-κB) and necroptosis signals (RIPK1, RIPK3 and MLKL) in kidney of AKI mouse. Notably, the co-culture system proved that Mincle in macrophage can aggravate the inflammation and necroptosis of mTEC induced by LPS, and artesunate suppressed the expression of Mincle in macrophage of kidney in AKI mouse. Overexpression of Mincle in BMDM restored the damage and necroptosis inhibited by artesunate in mTEC, indicating Mincle in macrophage is the target of artesunate to protect tubule cells in AKI. Our findings demonstrated that artesunate can significantly improve renal function in AKI, which may be related to the inhibition of Mincle-mediated macrophage inflammation, thereby reducing the damage and necroptosis to tubular cells that provide new option for the treatment of AKI.

LncRNA GAS5 protects against TGF-ß-induced renal fibrosis via the Smad3/miRNA-142-5p axis.

Increasing evidence shows that long noncoding RNAs (lncRNAs) play an important role in kidney disease. In this study, we investigated the role of the lncRNA growth arrest-specific 5 (GAS5) in the pathogenesis of renal fibrosis. We found that GAS5 was markedly decreased in the fibrotic kidney of a unilateral ureteral obstructive nephropathy mouse model. In addition, GAS5 was expressed in mouse tubular epithelial cells (mTECs) and interstitial fibroblasts in normal renal tissue and was especially highly expressed in the cytoplasm. In vitro experiments showed that GAS5 was downregulated by transforming growth factor-ß1 (TGF-ß1) in a dose- and time-dependent manner. Overexpression of GAS5 blocked TGF-ß1-induced collagen type I and fibronectin expression and vice versa. Mechanistic experiments revealed that Smad3 but not Smad2 drove the regulation of GAS5. More importantly, GAS5 interacted with miR-142-5p and was involved in the renoprotective effect by participating in the competing endogenous RNA network. Finally, we also found that knockdown of GAS5 promoted TGF-ß1-induced mouse tubular epithelial cell apoptosis via the Smad3 pathway. Taken together, our results uncovered a lncRNA/miRNA competing endogenous RNA network-based mechanism that modulates extracellular matrix formation and cell apoptosis via the Smad3 pathway.NEW & NOTEWORTHY In this work, we mainly discuss long noncoding RNA growth arrest-specific 5 (GAS5), acting in a renoprotective role via the Smad3/miRNA-142-5p axis, that modulates extracellular matrix formation and cell apoptosis. Overexpression of GAS5 effectively blocked renal fibrosis in vitro. This study reveals that GAS5 may represent as a novel and precision therapeutic target for alleviating renal fibrosis.

BAY61-3606 protects kidney from acute ischemia/reperfusion injury through inhibiting spleen tyrosine kinase and suppressing inflammatory macrophage response.

Acute kidney injury (AKI) is a highly prevalent clinical syndrome with high mortality and morbidity. Previous studies indicated that inflammation promotes tubular damage and plays a key role in AKI progress. Spleen tyrosine kinase (Syk) has been linked to macrophage-related inflammation in AKI. Up to date, however, no Syk-targeted therapy for AKI has been reported. In this study, we employed both cell model of LPS-induced bone marrow-derived macrophage (BMDM) and mouse model of ischemia/reperfusion injury (IRI)-induced AKI to evaluate the effects of a Syk inhibitor, BAY61-3606 (BAY), on macrophage inflammation in vitro and protection of kidney from AKI in vivo. The expression and secretion of inflammatory cytokines, both in vitro and in vivo, were significantly inhibited even back to normal levels by BAY. The upregulated serum creatinine and blood urea nitrogen levels in the AKI mice were significantly reduced after administration of BAY, implicating a protective effect of BAY on kidneys against IRI. Further analyses from Western blot, immunofluorescence staining and flow cytometry revealed that BAY inhibited the Mincle/Syk/NF-κB signaling circuit and reduced the inflammatory response. BAY also inhibited the reactive oxygen species (ROS), which further decreased the formation of inflammasome and suppressed the mature of IL-1ß and IL-18. Notably, these inhibitory effects of BAY on inflammation and inflammasome in BMDM were significantly reversed by Mincle ligand, trehalose-6,6-dibehenate. In summary, these findings provided compelling evidence that BAY may be an efficient inhibitor of the Mincle/Syk/NF-κB signaling circuit and ROS-induced inflammasome, which may help to develop Syk-inhibitors as novel therapeutic agents for AKI.

Identification of Hub Genes Associated with the Development of Acute Kidney Injury by Weighted Gene Co-Expression Network Analysis.

BACKGROUND: Acute kidney injury (AKI) is a severe clinical syndrome, causing a profound medical and socioeconomic burden worldwide. This study aimed to explore underlying molecular targets related to the progression of AKI. METHODS: A public database originated from the NCBI GEO database (serial number: GSE121190) and a well-established and unbiased method of weighted gene co-expression network analysis (WGCNA) to identify hub genes and potential pathways were used. Furthermore, the unbiased hub genes were validated in 2 classic models of AKI in a rodent model: chemically established AKI by cisplatin- and ischemia reperfusion-induced AKI. RESULTS: A total of 17 modules were finally obtained by the unbiased method of WGCNA, where the genes in turquoise module displayed strong correlation with the development of AKI. In addition, the results of gene ontology revealed that the genes in turquoise module were involved in renal injury and renal fibrosis. Thus, the hub genes were further validated by experimental methods and primarily obtained Rplp1 and Lgals1 as key candidate genes related to the progression of AKI by the advantage of quantitative PCR, Western blotting, and in situ tissue fluorescence. Importantly, the expression of Rplp1 and Lgals1 at the protein level showed positive correlation with renal function, including serum Cr and BUN. CONCLUSIONS: By the advantage of unbiased bioinformatic method and consequent experimental verification, this study lays the foundation basis for the pathogenesis and therapeutic agent development of AKI.

Renoprotective Effect of Oridonin in a Mouse Model of Acute Kidney Injury via Suppression of Macrophage Involved Inflammation.

Ischemia-reperfusion injury (IRI) is the major cause of acute kidney injury (AKI). The previous studies demonstrated that Oridonin can protect kidney against IRI-induced AKI, but the underlying molecular mechanism is unclear. In this study, it showed that Oridonin significantly improved kidney damage, and inhibited the expression of interleukin (IL)-1ß, IL-6, tumor necrosis factor (TNF)-α and MCP-1, as well as macrophage marker F4/80 in kidney and the secretion of inflammatory cytokins in serum of AKI mice in vivo. In addition, Oridonin also effectively reduced the expression and secretion of lipopolysaccharide (LPS)-induced inflammatory factors in macrophage cell line RAW264.7 in vitro. Notably, Oridonin strongly downregulated Mincle and AKT/nuclear factor-kappaB (NF-κB) signaling both in vivo and in vitro, and the results of cellular recovery experiments of overexpression of Mincle in macrophage suggested that Oridonin suppressed inflammatory response of macrophage through inhibiting Mincle, which may be the underlying mechanism of Oridonin improving injury in kidney of AKI mice. In summary, the above results indicated that Oridonin can protect kidney from IRI-induced inflammation and injury by inhibiting the expression of Mincle in macrophage.

Nodakenin alleviates renal ischaemia-reperfusion injury via inhibiting reactive oxygen species-induced NLRP3 inflammasome activation.

BACKGROUND: Acute kidney injury (AKI) is a vital contributor to chronic kidney disease and limited therapeutic options are existed to preserve the renal injury. The research presented here investigated the protective effect of nodakenin against AKI and the underlying mechanism. METHODS: The effect of nodakenin was investigated in ischaemia reperfusion-induced renal injury (IRI) of AKI mice and hypoxia-treated primary renal tubular cells. Briefly, renal functions including creatinine and urea nitrogen were determined and mechanisms associated inflammation were investigated by the advantage of immunohistochemistry, western blot, RT-PCR and flow cytometry. RESULTS: Deterioration of renal functions including and creatinine, urea nitrogen and tubular necrosis were observed in IRI-AKI model. In contrast, nodakenin strikingly alleviated the deterioration of creatinine, urea nitrogen and tubular necrosis when compared with IRI model. Moreover, nodakenin could significantly inhibit the expression of pro-inflammatory cytokines including interleukin (IL)-1ß, IL-6 and tumour necrosis factor-α both in hypoxia-treated primary renal tubular cells and in AKI model. Mechanistic studies revealed that nodakenin dramatically suppressed the production of reactive oxygen species and subsequent NLPR3 inflammasome activation. CONCLUSION: In summary, these findings provided a solid evidence base and a new drug option for the treatment of AKI.

Protocatechualdehyde attenuates obstructive nephropathy through inhibiting lncRNA9884 induced inflammation.

Persistent chronic inflammation and fibrosis product accumulation aggravate tubulointerstitial fibrosis (TIF), leading to the progression of chronic kidney disease. The aim of this study was designed to investigate the effect of protocatechualdehyde (PCA), a natural phenolic acid compound isolated from Salvia miltiorrhiza, on the unilateral ureteral obstruction (UUO)-induced fibrosis and inflammation and to elucidate the underlying mechanism in primary renal tubular epithelial cells (TECs). Results from the histology suggested that the moderate to severe deteriorations of renal dysfunction and the pathological changes in UUO could be relieved by PCA treatment. Mechanistic studies revealed that the effect of PCA was associated with the downregulation of Smad3 and NF-κB driven fibrosis and inflammation respectively. It is worth noting that PCA could inhibit the aberrant expression of inflammation cytokines such as iNOS, MCP-1, TNF-α in UUO, and IL-1ß-treated TECs. In addition, PCA also suppressed the expression of Smad3-dependent long noncoding RNA (lncRNA), 9884. Importantly, when overexpressing of lncRNA9884 in TECs by transfection of pcDNA3.1-lncRNA9884 plasmid, it revealed significant reversal of protein expression levels as that observed with only PCA, suggesting that PCA inhibits inflammation by mediating lncRNA9884/MCP-1 signaling pathway. Collectively, the current study establishes a foundational basis for PCA in future treatment of obstructive nephropathy.

Decursin inhibits the growth of HeLa cervical cancer cells through PI3K/Akt signaling.

Decursin, a coumarin compound isolated from Angelica gigas has been shown to possess multiple anti-tumor activities. But it's still little known about the effects associated with cervical cancer. To explore the anti-tumor role of decursin and gain insights into its underlying mechanisms, we analyzed proliferation in parallel with apoptosis and migration in HeLa cells. Our findings implied that decursin can provoke apoptosis, and inhibit cell proliferation, migration in HeLa cells. More importantly, decursin also inhibited the tumor growth in vivo. The mechanisms may be associated with the regulation of Akt activation, with implications for novel therapeutic strategies on cervical cancer.[Formula: see text].

Nodakenin alleviated obstructive nephropathy through blunting Snail1 induced fibrosis.

Tubulointerstitial fibrosis plays an important role in end-stage renal failure, and there are only limited therapeutic options available to preserve organ function. In the present study, we identified that nodakenin, a coumarin isolated from the roots of Angelicae gigas, functions effectively against unilateral ureteral obstruction (UUO)-induced fibrosis via down-regulating Snail1 expression. We established UUO-induced renal fibrosis in mice and then administered with nodakenin orally ata a dose of 1 and 10 mg/kg. The in-vivo results indicated that nodakenin protected obstructive nephropathy through its anti-inflammatory and anti-fibrotic properties. Nodakenin prevented the infiltration of inflammatory cells, alleviated the levels of pro-inflammatory cytokines, reduced the polarization of macrophages and down-regulating the aberrant deposition of extracellular matrix at the site of injury. Of note, nodakenin dramatically impeded Smad3, NF-κB p65 phosphorylation and Snail1 expression. In line with in vivo studies, nodakenin suppressed the expression of Snail1, Smad3 phosphorylation and fibrogenesis in TGF-ß1-treated renal epithelial cells in-vitro. Furthermore, we found that the effect of nodaknin against fibrosis was reversed in Snail1 overexpressing cells, whereas nodakenin could not further reduce expression of fibrogenesis in Snail1 silenced cells, suggesting that nodaknein may function through a Snail1-dependent manner. Collectively, this study reveal a critical role of nodakenin in the cure of renal fibrosis.

Oridonin Alleviates IRI-Induced Kidney Injury by Inhibiting Inflammatory Response of Macrophages via AKT-Related Pathways.

BACKGROUND Acute kidney injury (AKI) is one of the most common complications in clinic, but there is still no effective treatment. Oridonin, extracted from Rabdosia rubescens, has been identified to promote inhibitory effects on tumor, inflammatory and fibrosis by previous study. This study aimed to assess the kidney-protective role of Oridonin in AKI and the underlying mechanism by which Oridonin improves AKI in vivo and inhibits inflammation in LPS-induced bone marrow-derived macrophages (BMDM) in vitro. MATERIAL AND METHODS SPF C57BL/6J male mice (8 - 10 weeks old, body weight 20 - 25 g) were divided into 3 groups - sham group, AKI group, and Oridonin-treated AKI group - with 6 mice in each group. In the in vitro study, LPS-induced inflammatory BMDM cells were treated with Oridonin and agonist of AKT. The expression and secretion levels of inflammation-related indicators and AKT-related signaling molecules were detected by real-time PCR, ELISA, Western blot, and immunofluorescence. Also, various methods are used to assess renal function and pathological changes. RESULTS The results showed that Oridonin treatment significantly improved the serum creatinine and BUN levels in AKI mice. Interestingly, treatment with Oridonin also resulted in decreased the infiltration of macrophages in renal tissues of AKI mice, which was associated with decreased expression and activation of AKT and its related signaling pathways, such as NF-kappaB and STAT3, suggesting that Oridonin attenuates AKI kidney injury via a mechanism associated with reducing the inflammatory response of macrophages in the AKI kidney. This was investigated in vitro in macrophages, and the results showed that Oridonin reduced the LPS-stimulated inflammatory response in macrophages. Mechanistically, the addition of Oridonin reversed LPS-induced downregulation of AKT, NF-kappaB, and STAT3 expression and inflammatory response in macrophages, suggesting that Oridonin has a protective role, via the AKT-related signaling pathways, in reducing the inflammatory response of macrophages in AKI mice. This was further confirmed by adding agonist of AKT of IGF-1 to block the inhibitory effect of Oridonin on inflammatory response in vitro. CONCLUSIONS Oridonin ameliorates AKI kidney injuries by suppressing AKT-mediated inflammatory response of macrophages.

Quercetin protects against cisplatin-induced acute kidney injury by inhibiting Mincle/Syk/NF-κB signaling maintained macrophage inflammation.

Acute kidney injury (AKI) with high incidence and mortality is the main cause of chronic kidney disease. Previous studies have indicated that quercetin, an abundant flavonoid in plants, exhibited renoprotective role in AKI. However, the underlying mechanism is largely unknown. In this study, we try to explore whether quercetin protects against AKI by inhibiting macrophage inflammation via regulation of Mincle/Syk/NF-κB signaling. The results demonstrated that quercetin can significantly inhibit expression and secretion of IL-1ß, IL-6, and TNF-α in LPS-induced bone marrow-derived macrophages (BMDMs) and reduce activity of Mincle/Syk/NF-κB signaling in vitro. We also found that quercetin can strongly reduce the concentration of serum creatinine, BUN, IL-1ß, IL-6, and TNF-α in cisplatin-induced AKI model. Furthermore, quercetin down-regulated protein levels of Mincle, phosphorylated Syk and NF-κB in kidney macrophages of AKI, as well as inhibited M1, up-regulated M2 macrophage activity. Notably, the down-regulation of LPS-induced inflammation by quercetin was reversed after adding TDB (an agonist of Mincle) in BMDMs, suggesting that quercetin suppresses macrophage inflammation may mainly through inhibiting Mincle and its downstream signaling. In summary, these findings clarified a new mechanism of quercetin improving AKI-induced kidney inflammation and injury, which provides a new drug option for the clinical treatment of AKI.

A simple and highly purified method for isolation of glomeruli from the mouse kidney.

Highly purified mouse glomeruli are of great value for studying glomerulus-associated kidney diseases. Here, we developed a simple and rapid procedure for mouse glomerular isolation with large quantity and high purity based on the combination of size-selective sieving and differential adhesion techniques, which we termed the "differential adhesion method." In this method, mouse renal cortices were minced and digested with collagenase. Glomeruli were disassociated from tubules by successive sieving through 105-, 75-, and 40-µm cell strainers. The retained glomeruli-rich preparation on the 40-µm strainer was rinsed into a cell culture dish to allow tubules to adhere quickly to the dish while leaving most glomeruli floating (termed "differential adhesion"). The floating glomerular fraction was then subjected to another wash through the 40-µm strainer followed by an additional differential adhesion step to obtain highly purified glomeruli with yields of 8,357 ± 575 and purity of 96.1 ± 1.8% from one adult C57BL/6 mouse. The purity of the isolated glomeruli was further confirmed by high expression of the podocyte marker nephrin without detectable tubular marker cadherin-16. Importantly, we also found that although both the quantity and purity of the isolated glomeruli by this and the established Dynabeads method were comparable, glomeruli isolated by the current method showed much less inflammatory stress in terms of proinflammatory cytokine expression than the Dynabeads method. In conclusion, we established a newly mouse glomerular isolation method that is simple, rapid, cost effective, and productive. It provides an advanced methodology for research into glomerulus-related kidney diseases in the mouse.

Calycosin Ameliorates Diabetes-Induced Renal Inflammation via the NF-κB Pathway In Vitro and In Vivo.

BACKGROUND Diabetic nephropathy (DN), which is one of the primary causes of end-stage renal disease (ESRD), is increasingly diagnosed in patients due to the continuous increase in the prevalence of diabetic mellitus (DM). Astragali Radix, a traditional Chinese herb, is widely administrated to ameliorate the symptoms of diabetes and diabetic nephropathy, but its mechanism is still not yet fully defined. Calycosin (C16H12O5) is the major active component of Astragali Radix. In this study, we analyzed the role of calycosin in diabetic nephropathy and explored its underlying mechanism. MATERIAL AND METHODS Cell activation, inflammatory cytokines expression and secretion, and protein levels were analyzed in cultured mouse tubular epithelial cells (mTEC). db/db mice were intraperitoneally injected with 10 mg/(kg·d) calycosin or control saline for 4 weeks, followed by analysis of structure injury, inflammation, and NF-κB signaling activity. RESULTS Our results indicated that TNF-α and IL-1ß were significantly induced by advanced glycation end-products (AGEs), but calycosin remarkably reduced the expression of TNF-α and IL-1ß in the cultured mouse tubular epithelial cells (mTEC). Calycosin effectively alleviated kidney injury in diabetic kidneys of db/db mice during the progression of diabetic renal injury, indicated by the reduction of histological injury and immunohistochemical of inflammatory cytokines. Mechanistically, we identified calycosin inhibited diabetes-induced inflammation in kidneys by suppressing the phosphorylation of IKBa and NF-κB p65 in vitro and in vivo. CONCLUSIONS Calycosin significantly ameliorated diabetes-induced renal inflammation in diabetic renal injury by inhibition of the NF-κB-dependent signaling pathway in vivo and in vitro.

An optimized 5/6 nephrectomy mouse model based on unilateral kidney ligation and its application in renal fibrosis research.

5/6 Nephrectomy (PNx) on rat and mouse mimics renal failure after loss of kidney function in human, and it has been widely used in CKD researches. However, existing methods for PNx model construction present high mortality of animals after modeling due to hemorrhage and infection in or after surgery. Here, we report a novel and highly efficient PNx modeling method to simulate conventional 5/6 nephrectomy, which significantly reduced the mortality of animals and simplified the modeling procedures. In this novel modeling method, we directly ligated the upper and lower poles of left kidney after removal the right kidney 1 week later (l-PNx), which leads to necrosis of ligated upper and lower poles of the kidney and mimics the conventional 5/6 nephrectomy (c-PNx). After modeling 4 and 12 weeks, the serum creatinine, BUN and proteinuria levels were strongly increased in both c-PNx and l-PNx model. Importantly, compared with the c-PNx, l-PNx model present more severe renal fibrosis estimated by Masson staining, IHC and western blotting. The results showed that the protein levels of α-SMA were significantly increased in the kidney of c-PNx and l-PNx models, but more increase was found in l-PNx model. It is noteworthy that, compared with c-PNx model, the survival rate of l-PNx model was markedly increased. In summary, we established a novel and efficient 5/6 nephrectomy model, which can mimic conventional 5/6 nephrectomy to construct a renal fibrosis and renal failure mouse model, that is conducive to mechanism and treatment researches of CKD.

Specific Inhibitor of Smad3 (SIS3) Attenuates Fibrosis, Apoptosis, and Inflammation in Unilateral Ureteral Obstruction Kidneys by Inhibition of Transforming Growth Factor ß (TGF-ß)/Smad3 Signaling.

BACKGROUND Fibrosis is the common pathological feature in most kinds of chronic kidney disease (CKD). TGF-ß/Smads signaling is the master pathway regulating kidney fibrosis pathogenesis, in which Smad3 acts as the integrator of various pro-fibrosis signals. In this study, we analyzed the role of SIS3, a specific inhibitor of Smad3, in mouse unilateral ureteral obstruction (UUO) kidneys. MATERIAL AND METHODS UUO mice were intraperitoneally injected with 0.2 mg/kg/day or 2 mg/kg/day of SIS3 or control saline for 7 days, followed by analysis of structure injury, fibrosis status, inflammation, apoptosis, and TGF-ß/Smads signaling activity. RESULTS Our results indicated that SIS3 treatment dosage-dependently relieved the gross structure injury and tubular necrosis in UUO kidneys. Masson staining, immunohistochemistry, and real-time PCR showed significantly decreased extracellular matrix deposition, fibronectin staining intensity, and RNA levels of collagen I and collagen III in SIS3-treated UUO kidneys. SIS3 treatment also suppressed the activation of myofibroblasts, as evidenced by decreased expression levels of a-SMA and vimentin in UUO kidneys. The TGF-ß/Smads signaling activity analysis showed that SIS3 inhibited the phosphorylation of Smad3 but not Smad2 and decreased the protein level of TGF-ß1, suggesting specific inhibition of the TGF-ß/Smad3 pathway in UUO kidneys. Furthermore, SIS3 treatment also ameliorated the increased pro-inflammatory TNF-α and COX2 in UUO kidneys and circulating IL-1ß in UUO mice, and inhibited caspase-3 activity and the number of apoptotic cells. CONCLUSIONS SIS3 ameliorated fibrosis, apoptosis, and inflammation through inhibition of TGF-b/Smad3 signaling in UUO mouse kidneys.

Decursin inhibits the growth of HepG2 hepatocellular carcinoma cells via Hippo/YAP signaling pathway.

Targeted therapy has a pivotal role for the treatment of liver cancer. The aim of this current study was to examine the effects of decursin on the growth of HepG2 cells and the underlying mechanisms. Our present study showed that treatment of HepG2 cells with decursin significantly inhibited the growth of HepG2 cells by suppressing cell proliferation, cell cycle arresting, and promoting apoptosis in a dose- and time-dependent manner. Most significantly, administration of decursin dramatically impeded in vivo tumor growth in nude mice. Mechanically, it is noteworthy that decursin treatment provoked degradation of YAP by upregulating the expression of phosphorylated LATS1 and ßTRCP. Moreover, apoptosis caused by decursin could be reversed by a selective MST1/2 inhibitor, XMU-MP-1, suggesting that decursin may function through Hippo/YAP signaling. This study has identified that decursin is a potential agent for HCC therapy, and further research should be undertaken to facilitate its therapeutic application.