Mouse Model Of Unilateral Ureteral Obstruction Research Articles

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−β) to evaluate the anti-fibrotic effects of wogonin. The results demonstrated that wogonin treatment effectively attenuated TGF-β-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-β), 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.

Antisense oligonucleotide-mediated terminal intron retention of endoglin: A potential strategy to inhibit renal interstitial fibrosis

TGF-β is considered an important cytokine in the development of interstitial fibrosis in chronic kidney disease. The TGF-β co-receptor endoglin (ENG) tends to be upregulated in kidney fibrosis. ENG has two membrane bound isoforms generated via alternative splicing. Long-ENG was shown to enhance the extent of renal fibrosis in an unilateral ureteral obstruction mouse model, while short-ENG inhibited renal fibrosis. Here we aimed to achieve terminal intron retention of endoglin using antisense-oligo nucleotides (ASOs), thereby shifting the ratio towards short-ENG to inhibit the TGF-β1-mediated pro-fibrotic response. We isolated mRNA from kidney biopsies of patients with chronic allograft disease (CAD) (n = 12) and measured total ENG and short-ENG mRNA levels. ENG mRNA was upregulated 2.3 fold (p < 0.05) in kidneys of CAD patients compared to controls, while the percentage short-ENG of the total ENG mRNA was significantly lower (1.8 fold; p < 0.05). Transfection of ASOs that target splicing regulatory sites of ENG into TK173 fibroblasts led to higher levels of short-ENG (2 fold; p < 0.05). In addition, we stimulated these cells with TGF-β1 and measured a decrease in upregulation of ACTA2, COL1A1 and FN1 mRNA levels, and protein expression of αSMA, collagen type I, and fibronectin. These results show a potential for ENG ASOs as a therapy to reduce interstitial fibrosis in CKD.

Abstract 2969: Targeting novel senescence markers by conditionally active biologics eliminates senescence-associated secretory phenotype in in vitro and in vivo models

Abstract Clearing senescent cells has been shown to have promising effects against cancer and age-related pathologies in preclinical models as well as early human clinical trials. However, current senescent cell elimination strategies are focused on targets that do not distinguish between hyper-inflamed or SASP senescent cells versus relatively non-inflamed senescent cells. The use of senolytics in the clinic is still limited due to their cytotoxicity to either normal cells or potentially beneficial senescent cells. A more effective senescence cell targeted approach is needed to reduce side effects and increase the efficiency of senescence cell removal or reduction. Conditionally Active Biologics (CAB) technology is a proprietary platform unique in its ability to be selectively active in the context of diseased tissues, but not normal tissues. The aberrant accumulation of senescent cells in aged and cancerous tissue triggers inflammatory signaling through a senescence-associated secretory phenotype (SASP), promoting aging and tumor progression. Since our CAB technology is currently being tested as a cancer therapy by targeting cell surface markers such as AXL, ROR2, and CTLA4 in clinical studies, we explored whether CAB technology allows selective removal of senescent cells in SASP-associated microenvironments. In our previous report, we demonstrated several novel senescence-specific surface antigens upregulated in senescent cells (1). CAB antibodies targeting these senescence markers have no or very low binding to the target antigen on senescence cells in physiological, alkaline conditions, but have strong binding in glycolytic, acidic SASP conditions in in vitro binding assays. In in vitro antibody-dependent cell-mediated cytotoxicity assays, CAB antibodies were more potent against senescent cells compared to proliferating cells and displayed high selectivity for the glycolytic, acidic condition. To study whether CAB antibody reduces senescent cell occurrence in vivo, we developed a unilateral ureteral obstruction (UUO) mouse model to induce senescent cells, fibrosis, and inflammation in mouse kidney. The mice treated with CAB antibodies had a significant reduction of senescent cells and inflammatory cells infiltrated in the renal cortex, compared to the mice treated with benchmark and isotype antibodies in UUO kidneys. Thus, the CAB antibody-based therapeutic is a promising strategy for senescent cell removal in an inflammatory microenvironment. In conclusion, CAB technology provides a new generation of biologics with an increased safety margin and therapeutic index targeting SASP, acidic senescence cells in cancer and age-related diseases. (1). Chen J, Wang J, Liu H, Chang C, Boyle WJ, and Short JM. Abstract 4795: Conditionally active biologics eliminates senescence cells in cancer and aging. Cancer Res (2023) 83. Citation Format: Jian Chen, Jian Wang, Haizheng Liu, Cathy Chang, William J. Boyle, Jay M. Short. Targeting novel senescence markers by conditionally active biologics eliminates senescence-associated secretory phenotype in in vitro and in vivo models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2969.

FTO-mediated m6A mRNA demethylation aggravates renal fibrosis by targeting RUNX1 and further enhancing PI3K/AKT pathway.

Chronic kidney disease (CKD) is a global health burden, with ineffective therapies leading to increasing morbidity and mortality. Renal interstitial fibrosis is a common pathway in advanced CKD, resulting in kidney function and structure deterioration. In this study, we investigate the role of FTO-mediated N6-methyladenosine (m6A) and its downstream targets in the pathogenesis of renal fibrosis. M6A modification, a prevalent mRNA internal modification, has been implicated in various organ fibrosis processes. We use a mouse model of unilateral ureteral obstruction (UUO) as an invivo model and treated tubular epithelial cells (TECs) with transforming growth factor (TGF)-β1 as invitro models. Our findings revealed increased FTO expression in UUO mouse model and TGF-β1-treated TECs. By modulating FTO expression through FTO heterozygous mutation mice (FTO+/- ) invivo and small interfering RNA (siRNA) invitro, we observed attenuation of UUO and TGF-β1-induced epithelial-mesenchymal transition (EMT), as evidenced by decreased fibronectin and N-cadherin accumulation and increased E-cadherin levels. Silencing FTO significantly improved UUO and TGF-β1-induced inflammation, apoptosis, and inhibition of autophagy. Further transcriptomic assays identified RUNX1 as a downstream candidate target of FTO. Inhibiting FTO was shown to counteract UUO/TGF-β1-induced RUNX1 elevation invivo and invitro. We demonstrated that FTO signaling contributes to the elevation of RUNX1 by demethylating RUNX1 mRNA and improving its stability. Finally, we revealed that the PI3K/AKT pathway may be activated downstream of the FTO/RUNX1 axis in the pathogenesis of renal fibrosis. In conclusion, identifying small-molecule compounds that target this axis could offer promising therapeutic strategies for treating renal fibrosis.

C/EBPα aggravates renal fibrosis in CKD through the NOX4-ROS-apoptosis pathway in tubular epithelial cells

BackgroundChronic kidney disease (CKD) is a prevalent renal disorder with various risk factors. Emerging evidence indicates that the transcriptional factor CCAAT/enhancer binding protein alpha (C/EBPα) may be associated with renal fibrosis. However, the precise role of C/EBPα in CKD progression remains unexplored. MethodsWe investigated the involvement of C/EBPα in CKD using two distinct mouse models induced by folic acid (FA) and unilateral ureteral obstruction (UUO). Additionally, we used RNA sequencing and KEGG analysis to identify potential downstream pathways governed by C/EBPα. FindingsCebpa knockout significantly shielded mice from renal fibrosis and reduced reactive oxygen species (ROS) levels in both the FA and UUO models. Primary tubular epithelial cells (PTECs) lacking Cebpa exhibited reduced apoptosis and ROS accumulation following treatment with TGF-β. RNA sequencing analysis suggested that apoptosis is among the primary pathways regulated by C/EBPα, and identified NADPH oxidoreductase 4 (NOX4) as a key protein upregulated upon C/EBPα induction (ICCB280). Treatment with l-Theanine, a potential NOX4 inhibitor, mitigated renal fibrosis and inflammation in both the FA and UUO mouse models. InterpretationOur study unveils a role for C/EBPα in suppressing renal fibrosis, mitigating ROS accumulation, and reducing cell apoptosis. Furthermore, we investigate whether these protective effects are mediated by C/EBPα's regulation of NOX4 expression. These findings present a promising therapeutic target for modulating ROS and apoptosis in renal tubular cells, potentially offering an approach to treating CKD and other fibrotic diseases.

Noninvasive Assessment of Kidney Injury by Combining Structure and Function Using Artificial Intelligence-Based Manganese-Enhanced Magnetic Resonance Imaging.

Contrast-enhanced magnetic resonance imaging (MRI) is seriously limited in kidney injury detection due to the nephrotoxicity of clinically used gadolinium-based contrast agents. Herein, we propose a noninvasive method for the assessment of kidney injury by combining structure and function information based on manganese (Mn)-enhanced MRI for the first time. As a proof of concept, the Mn-melanin nanoprobe with good biocompatibility and excellent T1 relaxivity is applied in MRI of a unilateral ureteral obstruction mice model. The abundant renal structure and function information is obtained through qualitative and quantitative analysis of MR images, and a brand new comprehensive assessment framework is proposed to precisely identify the degree of kidney injury successfully. Our study demonstrates that Mn-enhanced MRI is a promising approach for the highly sensitive and biosafe assessment of kidney injury in vivo.

CXC chemokine receptor 7 ameliorates renal fibrosis by inhibiting β-catenin signaling and epithelial-to-mesenchymal transition in tubular epithelial cells

Renal fibrosis is a common feature of various chronic kidney diseases. However, the underlying mechanism remains poorly understood. The CXC chemokine receptor (CXCR) family plays a role in renal fibrosis; however, the detailed mechanisms have not been elucidated. In this study, we investigated the potential role of CXCR7 in mediating renal fibrosis. CXCR7 expression is decreased in unilateral ischemia–reperfusion injury (UIRI) and unilateral ureteral obstruction mouse models. Furthermore, CXCR7 was specifically expressed primarily in the Lotus Tetragonolobus Lectin-expressing segment of tubules, was slightly expressed in the peanut agglutinin-expressing segment, and was barely expressed in the Dolichos biflorus agglutinin-expressing segment. Administration of pFlag-CXCR7, an overexpression plasmid for CXCR7, significantly inhibited the activation of β-catenin signaling and protected against the progression of epithelial-to-mesenchymal transition (EMT) and renal fibrosis in a UIRI mouse model. Using cultured HKC-8 cells, we found that CXCR7 significantly downregulated the expression of active β-catenin and fibrosis-related markers, including fibronectin, Collagen I, and α-SMA. Furthermore, CXCR7 significantly attenuated TGF-β1-induced changes in β-catenin signaling, EMT and fibrosis. These results suggest that CXCR7 plays a crucial role in inhibiting the activation of β-catenin signaling and the progression of EMT and renal fibrosis. Thus, CXCR7 could be a novel therapeutic target for renal fibrosis.

Dual soluble epoxide hydrolase inhibitor - farnesoid X receptor agonist interventional treatment attenuates renal inflammation and fibrosis.

Renal fibrosis associated with inflammation is a critical pathophysiological event in chronic kidney disease (CKD). We have developed DM509 which acts concurrently as a farnesoid X receptor agonist and a soluble epoxide hydrolase inhibitor and investigated DM509 efficacy as an interventional treatment using the unilateral ureteral obstruction (UUO) mouse model. Male mice went through either UUO or sham surgery. Interventional DM509 treatment (10mg/kg/d) was started three days after UUO induction and continued for 7 days. Plasma and kidney tissue were collected at the end of the experimental protocol. UUO mice demonstrated marked renal fibrosis with higher kidney hydroxyproline content and collagen positive area. Interventional DM509 treatment reduced hydroxyproline content by 41% and collagen positive area by 65%. Renal inflammation was evident in UUO mice with elevated MCP-1, CD45-positive immune cell positive infiltration, and profibrotic inflammatory gene expression. DM509 treatment reduced renal inflammation in UUO mice. Renal fibrosis in UUO was associated with epithelial-to-mesenchymal transition (EMT) and DM509 treatment reduced EMT. UUO mice also had tubular epithelial barrier injury with increased renal KIM-1, NGAL expression. DM509 reduced tubular injury markers by 25-50% and maintained tubular epithelial integrity in UUO mice. Vascular inflammation was evident in UUO mice with 9 to 20-fold higher ICAM and VCAM gene expression which was reduced by 40-50% with DM509 treatment. Peritubular vascular density was reduced by 35% in UUO mice and DM509 prevented vascular loss. Interventional treatment with DM509 reduced renal fibrosis and inflammation in UUO mice demonstrating that DM509 is a promising drug that combats renal epithelial and vascular pathological events associated with progression of CKD.

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

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

The AMPK-Sirtuin 1-YAP axis is regulated by fluid flow intensity and controls autophagy flux in kidney epithelial cells

Shear stress generated by urinary fluid flow is an important regulator of renal function. Its dysregulation is observed in various chronic and acute kidney diseases. Previously, we demonstrated that primary cilium-dependent autophagy allows kidney epithelial cells to adapt their metabolism in response to fluid flow. Here, we show that nuclear YAP/TAZ negatively regulates autophagy flux in kidney epithelial cells subjected to fluid flow. This crosstalk is supported by a primary cilium-dependent activation of AMPK and SIRT1, independently of the Hippo pathway. We confirm the relevance of the YAP/TAZ-autophagy molecular dialog in vivo using a zebrafish model of kidney development and a unilateral ureteral obstruction mouse model. In addition, an in vitro assay simulating pathological accelerated flow observed at early stages of chronic kidney disease (CKD) activates YAP, leading to a primary cilium-dependent inhibition of autophagic flux. We confirm this YAP/autophagy relationship in renal biopsies from patients suffering from diabetic kidney disease (DKD), the leading cause of CKD. Our findings demonstrate the importance of YAP/TAZ and autophagy in the translation of fluid flow into cellular and physiological responses. Dysregulation of this pathway is associated with the early onset of CKD.

Asperulosidic Acid Ameliorates Renal Interstitial Fibrosis via Removing Indoxyl Sulfate by Up-Regulating Organic Anion Transporters in a Unilateral Ureteral Obstruction Mice Model

Asperulosidic acid is a bioactive iridoid isolated from Hedyotis diffusa Willd. with anti-inflammatory and renal protective effects. However, its mechanism on renal interstitial fibrosis has not been elucidated yet. The present study aims to explore whether asperulosidic acid could retard renal fibrosis by reducing the circulating indoxyl sulfate (IS), which is a uremic toxin and accelerates chronic kidney disease progression by inducing renal fibrosis. In this paper, a unilateral ureteral obstruction (UUO) model of Balb/C mice was established. After the mice were orally administered with asperulosidic acid (14 and 28 mg/kg) for two weeks, blood, liver and kidney were collected for biochemical, histological, qPCR and Western blot analyses. Asperulosidic acid administration markedly reduced the serum IS level and significantly alleviated the histological changes in glomerular sclerosis and renal interstitial fibrosis. It is noteworthy that the mRNA and protein levels of the organic anion transporter 1 (OAT1), OAT3 and hepatocyte nuclear factor 1α (HNF1α) in the kidney were significantly increased, while the mRNA expressions of cytochrome P450 2e1 (Cyp2e1) and sulfotransferase 1a1 (Sult1a1) in the liver were not altered after asperulosidic acid administration. These results reveal that asperulosidic acid could accelerate the renal excretion of IS by up-regulating OATs via HNF1α in UUO mice, thereby alleviating renal fibrosis, but did not significantly affect its production in the liver, which might provide important information for the development of asperulosidic acid.

Establishment and Application a Novel Reversible Unilateral Ureteral Obstruction (RUUO) Mouse Model and in Mechanism Study of AKI to CKD

Establishment and Application a Novel Reversible Unilateral Ureteral Obstruction (RUUO) Mouse Model and in Mechanism Study of AKI to CKD

Dose-Dependent Nephroprotective Effects of an ALK5 Inhibitor in the Unilateral Ureteral Obstruction (UUO) Mouse Model of Kidney Fibrosis

Dose-Dependent Nephroprotective Effects of an ALK5 Inhibitor in the Unilateral Ureteral Obstruction (UUO) Mouse Model of Kidney Fibrosis

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

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

Neuropilin 1 promotes unilateral ureteral obstruction-induced renal fibrosis via RACK1 in renal tubular epithelial cells.

Neuropilin 1 (NRP1) is a single-channel transmembrane glycoprotein whose role and mechanism in renal fibrosis remain incompletely elucidated. Therefore, we investigated the effect of NRP1 on renal fibrosis and its potential mechanism. NRP1 expression in the renal sections from patients with chronic kidney disease (CKD) and a unilateral ureteral obstruction (UUO) mouse model was detected. Nrp1 overexpression or knockdown plasmid was transfected into mice, TKPTS mouse kidney proximal tubular epithelial cells (TECs), and rat kidney fibroblasts, after which pathological injury evaluation and fibrosis marker detection were conducted. The direct interaction of the receptor of activated protein C kinase 1 (RACK1) with NRP1 was validated by immunoprecipitation and Western blot analysis. We found that the upregulated renal NRP1 expression in patients with CKD was located in proximal TECs, consistent with the degree of interstitial fibrosis. In the UUO mouse model, NRP1 expression was upregulated in the kidney, and overexpression of Nrp1 increased the mRNA and protein expression of fibronectin (Fn) and α-smooth muscle actin (α-SMA), whereas Nrp1 knockdown significantly reduced Fn and α-SMA expression and downregulated the inflammatory response. NRP1 promoted transforming growth factor β1 (TGF-β1)-induced profibrotic responses in the TKPTS cells and fibroblasts, and Nrp1 knockdown partially reversed these responses. Immunoprecipitation combined with liquid chromatography-tandem mass spectrometry verified that NRP1 can directly bind to RACK1, and Rack1 knockdown reversed the NRP1-induced fibrotic response. In summary, NRP1 may enhance the TGF-β1 pathway by binding to RACK1, thus promoting renal fibrosis.NEW & NOTEWORTHY Although a few studies have confirmed the correlation between neuropilin 1 (NRP1) and renal diseases, the mechanism of NRP1 in renal fibrosis remains unclear. Here, we investigated the effects of NRP1 on renal fibrosis through in vitro and in vivo experiments and explored the possible downstream mechanisms. We found that NRP1 can stimulate the TGF-β1 signaling pathway, possibly by binding to RACK1, thereby promoting renal fibrosis.

Losartan alleviates renal fibrosis by inhibiting the biomechanical stress-induced epithelial-mesenchymal transition of renal epithelial cells

Angiotensin receptor blockers (ARBs) have been reported to be beneficial of renal fibrosis, but the molecular and cellular mechanisms are still unclear. In this study, we investigated the effectiveness and relevant mechanism of ARBs in alleviating renal fibrosis, especially by focusing on biomechanical stress-induced epithelial to mesenchymal transition (EMT) of renal epithelial cells. Unilateral ureteral obstruction (UUO) renal fibrosis model was established in mice by ligating the left ureter, and then randomly received losartan at a low dose (1 mg/kg) or a regular dose (3 mg/kg) for 2 weeks. Compared to the control, histological analysis showed that losartan treatment at either a low dose or a regular dose effectively attenuated renal fibrosis in the UUO model. To further understand the mechanism, we ex vivo loaded primary human renal epithelial cells to 50 mmHg hydrostatic pressure. Western blot and immunostaining analyses indicated that the loading to 50 mmHg hydrostatic pressure for 24 h significantly upregulated vimentin, β-catenin and α-SMA, but downregulated E-cadherin in renal epithelial cells, suggesting the EMT. The addition of 10 or 100 nM losartan in medium effectively attenuated the EMT of renal epithelial cells induced by 50 mmHg hydrostatic pressure loading. Our in vivo and ex vivo experimental data suggest that losartan treatment, even at a low dose can effectively alleviate renal fibrosis in mouse UUO model, at least partly by inhibiting the biomechanical stress-induced EMT of renal epithelial cells. A low dose of ARBs may repurpose for renal fibrosis treatment.

Ecliptasaponin A attenuates renal fibrosis by regulating the extracellular matrix of renal tubular cells

Renal fibrosis is the most common manifestation of end-stage renal disease (ESRD), including diabetic kidney disease (DKD), but there is no effective treatment in renal fibrosis. Natural products are a rich source of clinical drug research and have been used in the clinical research of various diseases. In this study, we searched for traditional Chinese medicine monomers that attenuate fibrosis and assessed their effect on the fibrosis marker connective tissue growth factor (CTGF) in cells which we found ecliptasaponin A. Subsequently, we evaluated the effect of ecliptasaponin A on renal fibrosis in the classic renal fibrosis unilateral ureteral obstruction (UUO) mouse model and found that ecliptasaponin A could reduce the renal collagen fiber deposition and renal extracellular matrix (ECM) protein expression in UUO mice. In vitro, ecliptasaponin A can inhibit ECM protein expression in human kidney-2 (HK-2) cells induced by transforming growth factor-beta1 (TGFβ1). To further clarify the mechanism of ecliptasaponin A in attenuating renal fibrosis, we performed transcriptome sequencing of HK-2 cells treated with TGFβ1 and ecliptasaponin A. The functions and pathways were mainly enriched in the extracellular matrix and TGFβ signalling pathway. Matrix metalloproteinase 10 (MMP10) and matrix metalloproteinase 13 (MMP13) are the main differentially expressed genes in extracellular matrix regulation. Then, we measured MMP10 and MMP13 in the cells and found that ecliptasaponin A had a significant inhibitory effect on MMP13 expression but not on MMP10 expression. Furthermore, we overexpressed MMP13 in HK-2 cells treated with TGFβ1 and found that MMP13 promoted HK-2 cell injury. Our findings suggest that ecliptasaponin A can attenuate renal fibrosis, which may provide a new method for treating renal fibrosis clinically.

Connexin-43 hemichannels orchestrate NOD-like receptor protein-3 (NLRP3) inflammasome activation and sterile inflammation in tubular injury

BackgroundWithout a viable cure, chronic kidney disease is a global health concern. Inflammatory damage in and around the renal tubules dictates disease severity and is contributed to by multiple cell types. Activated in response to danger associated molecular patterns (DAMPs) including ATP, the NOD-like receptor protein-3 (NLRP3) inflammasome is integral to this inflammation. In vivo, we have previously observed that increased expression of Connexin 43 (Cx43) is linked to inflammation in chronic kidney disease (CKD) whilst in vitro studies in human proximal tubule cells highlight a role for aberrant Cx43 hemichannel mediated ATP release in tubule injury. A role for Cx43 hemichannels in priming and activation of the NLRP3 inflammasome in tubule epithelial cells remains to be determined.MethodsUsing the Nephroseq database, analysis of unpublished transcriptomic data, examined gene expression and correlation in human CKD. The unilateral ureteral obstruction (UUO) mouse model was combined with genetic (tubule-specific Cx43 knockout) and specific pharmacological blockade of Cx43 (Peptide5), to explore a role for Cx43-hemichannels in tubule damage. Human primary tubule epithelial cells were used as an in vitro model of CKD.ResultsIncreased Cx43 and NLRP3 expression correlates with declining glomerular filtration rate and increased proteinuria in biopsies isolated from patients with CKD. Connexin 43-tubule deletion prior to UUO protected against tubular injury, increased expression of proinflammatory molecules, and significantly reduced NLRP3 expression and downstream signalling mediators. Accompanied by a reduction in F4/80 macrophages and fibroblast specific protein (FSP1+) fibroblasts, Cx43 specific hemichannel blocker Peptide5 conferred similar protection in UUO mice. In vitro, Peptide5 determined that increased Cx43-hemichannel activity primes and activates the NLRP3 inflammasome via ATP-P2X7 receptor signalling culminating in increased secretion of chemokines and cytokines, each of which are elevated in individuals with CKD. Inhibition of NLRP3 and caspase 1 similarly decreased markers of tubular injury, whilst preventing the perpetual increase in Cx43-hemichannel activity.ConclusionAberrant Cx43-hemichannel activity in kidney tubule cells contributes to tubule inflammation via activation of the NLRP3 inflammasome and downstream paracrine mediated cell signalling. Use of hemichannel blockers in targeting Cx43-hemichannels is an attractive future therapeutic target to slow or prevent disease progression in CKD.-uWW98vNU7-T3Uscg54sCsVideo

LMCD1 is involved in tubulointerstitial inflammation in the early phase of renal fibrosis by promoting NFATc1-mediated NLRP3 activation.

Prolonged renal inflammation contributes to fibrosis, which may eventually lead to irreversible chronic kidney disease. Our previous work demonstrated that LIM and cysteine-rich domain 1 (LMCD1) are associated with renal interstitial fibrosis in a 21-day unilateral ureteral obstruction (21UUO) mouse model. Interestingly, based on the gene expression omnibus database, we found that LMCD1 is enhanced in the mouse kidney as early as 5, 7, and 10days following unilateral ureteral obstruction (UUO), suggesting that LMCD1 may exert its function in an earlier phase. To validate this conjecture, a 7UUO mouse model and a tumor necrosis factor-α (TNF-α)-stimulated HK-2 cell model were established, followed by injection of adenovirus vectors carrying short hairpin RNA targeting LMCD1. LMCD1 silencing ameliorated renal collagen deposition and reduced the expression of profibrotic factors in the 7UUO model. LMCD1 silencing alleviated tubulointerstitial inflammation by mitigating F4/80+ cell infiltration, monocyte chemoattractant protein-1 release and nuclear factor-κB activation. In addition, LMCD1 silencing suppressed NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome activation and nuclear factor of activated T cells 1 (NFATc1) nuclear translocation. Consistent results were obtained in TNF-α-stimulated HK-2 cells in vitro. Mechanistically, the transcriptional coactivator LMCD1 cooperates with the transcription factor NFATc1 to increase NLRP3 expression. Collectively, these findings suggest that LMCD1 participates in tubulointerstitial inflammation via an LMCD1-NFATc1/NLRP3 mechanism. LMCD1 may therefore become a potential target for the control of renal inflammation and fibrosis.

Discovery and Design of Novel Cyclic Peptides as Specific Inhibitors Targeting CCN2 and Disrupting CCN2/EGFR Interaction for Kidney Fibrosis Treatment.

Kidney fibrosis is a serious consequence of chronic kidney disease (CKD), and currently, there is no effective pharmacological treatment available. Cellular communication network-2 (CCN2/CTGF) is an extracellular matrix (ECM) protein that regulates the fibrotic process by activating the epidermal growth factor receptor (EGFR) signaling pathway. We herein present the discovery and structure-activity relationship study of novel peptides targeting CCN2 to develop potent and stable specific inhibitors of the CCN2/EGFR interaction. Remarkably, the 7-mer cyclic peptide OK2 exhibited potent activities to inhibit CCN2/EGFR-induced STAT3 phosphorylation and cellular ECM protein synthesis. Subsequent in vivo studies demonstrated that OK2 significantly alleviated renal fibrosis in a unilateral ureteral obstruction (UUO) mouse model. Moreover, this study first revealed that the peptide candidate could efficiently block CCN2/EGFR interaction through binding to the CT domain of CCN2, providing a new alternative strategy for peptide-based targeting of CCN2 and modulating CCN2/EGFR-mediated biological functions in kidney fibrosis.