Unilateral Ureteral Obstruction Kidneys Research Articles

#1453 EZH2 is an epigenetic regulator of renal gluconeogenesis in chronic kidney disease through downregulation of PCK1

Abstract Background and Aims Impaired renal gluconeogenesis has been recently identified as a hallmark of chronic kidney diseases (CKD), and is tightly correlated with the progression of renal tubulointerstitial fibrosis. The methyltransferase enhancer of zeste homolog 2 (EZH2) is an epigenetic regulator playing an important role in renal tubulointerstitial fibrosis. Whether renal gluconeogenesis can be epigenetically regulated is currently unknown. In the current study, we studied EZH2-mediated epigenetic regulation of renal gluconeogenesis in three different mouse models of renal fibrosis. Method Mouse models of renal fibrosis were established by unilateral ureteral obstruction (UUO) operation, unilateral ischemia reperfusion injury or folic acid (FA) injection. Human proximal tubular cell line (HK2), primary tubular cells or primary tubules was used as an in vitro or ex vivo model. EZH2 was conditional knockout or inhibited by 3-DZNeP in mice. Inhibition of EZH2 by 3-DZNeP or overexpressed by adenovirus was performed in vitro. RNA sequencing and cleavage under targets and tagmentation (CUT&Tag) sequencing analysis were performed and followed by quantitative PCR, CUT&Tag and Western blotting analysis. Glucogenic metabolites were measured and systemic glucose metabolism was assessed by tolerance tests. Results By deletion of Ezh2 gene in mice through a ubiquitous expressed Cre/Esr1 system, we confirmed the pro-fibrotic effect of EZH2 in unilateral ureteral obstruction (UUO) kidneys. Through RNA sequencing and CUT&Tag sequencing analysis, we found that the phosphoenolpyruvate carboxykinase 1 (PCK1), a critical enzyme in gluconeogenesis, is negatively regulated by the methyltransferase EZH2 in fibrotic kidneys, which was further confirmed by quantitative PCR, CUT&Tag and Western blotting analysis in renal cells and UUO or FA kidneys. We further showed that pharmaceutical inhibition of EZH2 by 3-DZNeP enhances PCK1 expression in fibrotic kidneys of three mouse models. Moreover, the concentration of lactate, a glucogenic metabolic intermediate, was increased in mouse fibrotic kidneys, which was reduced by EZH2 inhibition. A further experiment showed that systemic glucose metabolism was impaired in UUO mice and restored by EZH2 inhibition as assessed by insulin or glucose tolerance test. Upregulated EZH2 in UUO kidneys is colocalized with the proximal tubular marker as shown by immunofluorescent staining. The direct anti-gluconeogenesis effect of EZH2 on renal proximal epithelial cells and primary renal tubules was shown by analysis of gluconeogenic gene expression and production of glucose or lactate in human HK2 cells treated with EZH2 inhibitor or exogeneous expression of EZH2 by adenovirus transfection. Importantly, we further confirmed the direct effect of tubular EZH2 on gluconeogenesis in tubular specific deleted EZH2 mice (Ezh2fl/fl;Cdh16-Cre). Moreover, inhibition of PCK1 by 3-mercaptopropionic acid abrogated the pro-gluconeogenic effect and anti-fibrotic effect of EZH2 inhibitor in renal cells and UUO kidneys. Finally, RNA sequencing data from kidney allograft biopsies showed that EZH2 is progressively increased in CKD patients. Conclusion We conclude that EZH2 promotes renal tubulointerstitial fibrosis through epigenetic inhibition of PCK1. Our study suggests that therapeutic restoration of the impaired renal gluconeogenesis is beneficial to CKD.

#2104 Neutrophil extracellular traps have a pivotal role in tubulointerstitial fibrosis

Abstract Background and Aims Tubulointerstitial fibrosis (TIF) is a typical pathological change in chronic kidney disease (CKD). However, the mechanisms underlying tubulointerstitial fibrosis are still to be fully elucidated. Neutrophil extracellular traps (NETs) participate in the pathophysiological processes of various kidney diseases, but their role in CKD-TIF has not been reported. Here, we investigated the role of NETs in tubulointerstitial fibrosis. Method Immunofluorescence was employed to assess the levels and localization of NETs in the renal tissues of CKD patients. The GSE66494 human CKD microarray dataset was explored to analyze the changes of PAD4 levels. Three CKD mice models, including 14d unilateral ureteral obstruction (UUO), 28d ischemia/reperfusion (I/R) injury, and adenine-induced models were constructed to detect the expression levels of NETs in the kidneys. Then, we established a NETs-deficient (PAD4−/−) CKD mice model and assessed changes in renal function, NETs levels, and renal fibrosis. In vitro, phorbol-12-myristate-13-acetate (the classic inducer of NETs) was used to induce the generation of NETs. Intervention was performed on rat fibroblast NRK49F cells to investigate the mechanism of TIF induced by NETs. Results We first identified NETs levels were significantly elevated in the renal tissues of CKD patients. Additionally, chip data results also indicated elevated PAD4 levels in the renal tissues of CKD patients. NETs levels were elevated in the kidneys of UUO, I/R, and adenine-induced CKD mice, with deposition in the renal interstitium. PAD4-deficient mice did not form neutrophil extracellular traps. Knocking out PAD4 alleviated tubulointerstitial fibrosis in UUO, adenine, and I/R mice after blocking NETs formation. In vitro, NETs intervention in fibroblasts can promote their activation and transformation into myofibroblasts. Conclusion The deletion of NETs by PAD4−/− could significantly improve CKD-TIF. The inhibition of NETs might provide a new molecular target for the prevention and treatment of CKD.

#1581 Anoctamin 3 inhibits gluconeogenesis and contributes to renal interstitial fibrosis through EZH2

Abstract Background and Aims Renal fibrosis is the common pathological pathway of various chronic kidney diseases progressing to the end stage of renal failure. Anoctamin 3 (ANO3) has been identified as a K-channel regulator and been extensively studied in the field of neurobiology. The role of ANO3 in kidney diseases is currently unknown. Method Mouse models of renal fibrosis were established by unilateral ureteral obstruction (UUO) operation or aristolochic acid I (AAI) injection. Human proximal tubular cell line (HK2) was used as an in vitro model. ANO3 was deleted in vitro by siRNA. Western blotting analysis was performed. Glucogenic metabolites were measured. Results We found that ANO3 is upregulated in mouse kidneys with unilateral ureteral obstruction (UUO) or aristolochic acid nephropathy (AAN), and tightly correlated with the progression of renal fibrosis and negatively correlated with expression of several rate-limiting enzymes of renal gluconeogenesis. Knockdown of ANO3 with siRNA increased the concentration of glucose and reduced the concentration of lactate in the supernatant of TGF-β stimulated renal proximal tubular (HK2) cells. In parallel, transfection of ANO3 siRNA significantly reduced the expression of epithelial-to-mesenchymal transition (EMT) and fibrotic markers in TGF-β stimulated HK2 cells. We further showed that ANO3 expression is positively correlated with the expression of a methyltransferase enhancer of zeste homologue 2 (EZH2) in UUO and AAN mouse kidneys by time. Transfection of ANO3 siRNA significantly reduced the expression of EZH2 in TGF-β stimulated HK2 cells. Overexpression of EZH2 by adenovirus reversed the pro-gluconeogenic and anti-fibrotic effect of ANO3 siRNA in TGF-β stimulated HK2 cells by measuring the concentration of glucose and lactate in supernatant and analyzing the expression of EMT or fibrotic markers. Conclusion In conclusion, ANO3 inhibits renal gluconeogenesis through EZH2 and contributes to renal fibrosis. ANO3 could be promising therapeutic target to inhibit renal fibrosis in chronic kidney disease patients.

557 Dual TGFβR1/MAP4K4 inhibitor reduces kidney injury in a mouse model of renal fibrosis.

OBJECTIVES/GOALS: Renal fibrosis is a critical pathophysiological event in chronic kidney diseases. Our goal is to determine the ability of dual-inhibitor of transforming growth factor beta receptor 1 (TGFα²R1) and mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4), TK850, on reducing kidney fibrosis. METHODS/STUDY POPULATION: To test the renal anti-fibrotic action ofdual TK850,8-10-week-old male and female C57BL/6mice with unilateral ureteral obstruction (UUO) induced kidney fibrosis were used. Mice were separated into 3 groups: group 1 contained mice that had UUO surgery (UUO control), group 2 contained mice prophylactically treated with TK850 thatstarted 7 days prior to UUO(UUO-P,20 mpk/d/ip), and group 3 contained mice interventionally treated with TK850 that started3 days after UUO(UUO-I,20 mpk/d/ip). Ten days following UUO the kidneys and blood were collected for analysis. Renal fibrosis was assessed from hydroxyproline content (measure of collagen)and histological collagen analysis using Picrosirius red stain. RESULTS/ANTICIPATED RESULTS: Renal hydroxyproline was increased equally in the UUO kidney of male (5.4 ± 0.41 µg/10mg, n=5) and female mice (5.5 ± 0.50 µg/10mg, n=5) compared to the contralateral control kidney (2.9 ± 0.14 µg/10mg, n=10). TK850 treatment in UUO-P mice (n=10, 3.4 ± 0.24 µg/10mg) and UUO-I mice (4.30 ± 0.20 µg/10mg, n=10) had significantly reduced hydroxyproline levels. Histopathological evaluation revealed that kidney injury increased collagen deposition in the UUO kidney (17.1 ± 0.43% collagen positive area, n=10) compared to the control kidney (2.0 ± 0.23%, n=10). TK850 treatment in UUO-P mice significantly attenuated collagen deposition (10.5 ± 0.38%, n=10), while UUO-I had significantly reduced collagen deposition as well (13.1 ± 0.25%, n=10). DISCUSSION/SIGNIFICANCE: Taken together, these results validate the dual TGFβR1/MAP4K4 inhibitor, TK850 as a potential therapeutic to mitigate renal fibrosis and supports the emergence of a combinational pharmacotherapeutic approach for multi-factorial kidney diseases.

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.

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

Epigenetic regulations, including DNA methylation, are critical to the development and progression of kidney fibrosis, but the underlying mechanisms remain elusive. Here, we show that fibrosis of the mouse kidney was associated with the induction of DNA methyltransferases and increases in global DNA methylation and was alleviated by the DNA methyltransferase inhibitor 5-Aza-2’-deoxycytidine (5-Aza). Genome wide analysis demonstrated the hypermethylation of 94 genes in mouse unilateral ureteral obstruction kidneys, which was markedly reduced by 5-Aza. Among these genes, Hoxa5 was hypermethylated at its gene promoter and this hypermethylation was associated with reduced HOXA5 expression in fibrotic mouse kidneys after ureteral obstruction or unilateral ischemia-reperfusion injury. 5-Aza prevented Hoxa5 hypermethylation, restored HOXA5 expression, and suppressed kidney fibrosis. Down-regulation of HOXA5 was verified in human kidney biopsies from patients with chronic kidney disease and correlated with the increased kidney fibrosis and DNA methylation. Kidney fibrosis was aggravated by conditional knockout of Hoxa5 and alleviated by conditional knockin of Hoxa5 in kidney proximal tubules of mice. Mechanistically, we found that HOXA5 repressed Jag1 transcription by directly binding to its gene promoter, resulting in the suppression of JAG1-NOTCH signaling during kidney fibrosis. Thus, our results indicate that loss of HOXA5 via DNA methylation contributes to fibrogenesis in kidney diseases by inducing JAG1 and consequent activation of the NOTCH signaling pathway.

Activation of the YAP/KLF5 transcriptional cascade in renal tubular cells aggravates kidney injury

The Hippo/YAP pathway plays a critical role in tissue homeostasis. Our previous work demonstrated that renal tubular YAP activation induced by double knockout of the upstream Hippo kinases Mst1 and Mst2 (Mst1/2 dKO) promotes tubular injury and renal inflammation under basal conditions. However, the importance of tubular YAP activation remains to be established in injured kidneys in which many other injurious pathways are simultaneously activated. Here, we show that tubular YAP was already activated 6 h after unilateral ureteral obstruction (UUO). Tubular YAP deficiency greatly attenuated tubular cell over-proliferation, tubular injury and renal inflammation induced by UUO or cisplatin. YAP promoted the transcription of the transcription factor KLF5. Consistently, the elevated expression of KLF5 and its target genes in Mst1/2 dKO or UUO kidneys was blocked by ablation of Yap in tubular cells. Inhibition of KLF5 prevented tubular cell over-proliferation, tubular injury and renal inflammation in Mst1/2 dKO kidneys. Therefore, our results demonstrate that tubular YAP is a key player in kidney injury. YAP and KLF5 form a transcriptional cascade, in which tubular YAP activation induced by kidney injury promotes KLF5 transcription. Activation of this cascade induces tubular cell over-proliferation, tubular injury and renal inflammation.

Alantolactone Attenuates Renal Fibrosis via Inhibition of Transforming Growth Factor β/Smad3 Signaling Pathway.

Renal fibrosis is characterized by the accumulation of extracellular matrix proteins and interstitial fibrosis. Alantolactone is known to exert anticancer, anti-inflammatory, antimicrobial and antifungal effects; however, its effects on renal fibrosis remains unknown. Here, we investigated whether alantolactone attenuates renal fibrosis in mice unilateral ureteral obstruction (UUO) and evaluated the effect of alantolactone on transforming growth factor (TGF) signaling pathway in renal cells. To evaluate the therapeutic effect of alantolactone, cell counting kit-8 (CCK-8) assay, histological staining, Western blot analysis, and real-time quantitative polymerase chain reaction were performed in UUO kidneys in vivo and in TGF-β-treated renal cells in vitro. Alantolactone (0.25 to 4 µM) did not affect the viability of renal cells. Mice orally administered 5 mg/kg of alantolactone daily for 15 days did not show mortality or liver toxicity. Alantolactone decreased UUO-induced blood urea nitrogen and serum creatinine levels. In addition, it significantly alleviated renal tubulointerstitial damage and fibrosis and decreased collagen type I, fibronectin, and α-smooth muscle actin (α-SMA) expression in UUO kidneys. In NRK-49F cells, alantolactone inhibited TGF-βstimulated expression of fibronectin, collagen type I, plasminogen activator inhibitor-1 (PAI-1), and α-SMA. In HK-2 cells, alantolactone inhibited TGF-β-stimulated expression of collagen type I and PAI-1. Alantolactone inhibited UUO-induced phosphorylation of Smad3 in UUO kidneys. In addition, it not only decreased TGF-β secretion but also Smad3 phosphorylation and translocation to nucleus in both kidney cell lines. Alantolactone improves renal fibrosis by inhibiting the TGF-β/Smad3 signaling pathway in obstructive nephropathy. Thus, alantolactone is a potential therapeutic agent for chronic kidney disease.

Vitexin attenuates chronic kidney disease by inhibiting renal tubular epithelial cell ferroptosis via NRF2 activation

BackgroundChronic kidney disease (CKD) involves a variety of pathological processes, and ferroptosis plays a vital role in CKD progression. Targeting ferroptosis is a promising strategy for the treatment of CKD. However, inhibitors of ferroptosis have not been used in the clinical treatment of CKD. Vitexin is a natural flavonoid with many biological activities and protective effects against various diseases. However, whether vitexin can prevent the progression of CKD is not known.MethodsIn vivo, the effect of vitexin on CKD was evaluated by using mouse models of unilateral ureteral obstruction (UUO) and unilateral ischemia–reperfusion (UIR). Western blotting, Sirius red staining and transmission electron microscopy were used to analyze renal tubular injury, interstitial fibrosis, and inflammation in the kidneys of UUO and UIR mice. In vitro, CCK8 assays and lipid peroxidation assays were performed to analyze cell viability and lipid peroxidation in human renal tubular epithelial cells (HK2 cells) induced by erastin. The activation of renal fibroblasts (NRK-49 F cells) was also analyzed. Additionally, an in-silico protein-drug docking model and coimmunoprecipitation were performed to determine the direct substrate of vitexin.ResultsIn vivo, vitexin treatment significantly ameliorated renal tubular injury, interstitial fibrosis, and inflammation in the kidneys of UUO and UIR mice. Additionally, our results showed that vitexin significantly attenuated UUO- and UIR-induced ferroptosis in renal tubular epithelial cells by upregulating glutathione peroxidase 4 (GPX4) protein levels and inhibiting lipid peroxidation in mouse kidneys. In vitro, treatment with vitexin inhibited erastin-induced ferroptosis in HK2 cells. Moreover, vitexin inhibited the expression of collagen I and α-SMA (alpha-smooth muscle actin) in NRK-49 F cells induced by the supernatant of erastin-treated HK2 cells. Mechanistically, our results suggested that vitexin could activate the NRF2/heme oxygenase-1 (HO-1) pathway by inhibiting the KEAP1- and ubiquitination-mediated degradation of NRF2, thereby increasing the expression of GPX4, and further inhibiting lipid peroxidation and ferroptosis. Additionally, knockout of NRF2 greatly inhibited the antiferroptotic effects of vitexin.ConclusionsTaken together, our results indicate that vitexin can protect against renal tubular epithelial cell ferroptosis in CKD by activating the KEAP1/NRF2/HO-1 pathway and is a promising drug to treat CKD.

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.

Minichromosome maintenance 6 protects against renal fibrogenesis by regulating DUSP6-mediated ERK/GSK-3β/Snail1 signaling

Minichromosome maintenance 6 (MCM6) has been implicated in the progression of various malignant tumors; however, its exact physiological function in kidney diseases remains unclear. Here, we demonstrated that MCM6 levels showed a significant increase in the proximal tubular cells during progressive renal fibrosis in two unrelated invivo fibrotic models, including unilateral ureteral obstruction (UUO) and unilateral ischemia-reperfusion injury (UIRI). Depletion of MCM6 aggravated partial epithelial-mesenchymal transition, extracellular matrix accumulation, and myofibroblast activation in the kidneys of UUO or UIRI mice. Conversely, overexpression of MCM6 promoted the recovery of E-cadherin and retarded UUO- or UIRI-induced renal fibrosis. In addition, DUSP6 expression substantially decreased in fibrotic kidneys, and it might be involved in MCM6-induced renal fibrosis by regulating the activation of ERK/GSK-3β/Snail1 signaling. In conclusion, our results highlight the significance of MCM6 in renal fibrosis, providing a potential therapeutic target for patients with chronic kidney disease.

Systemic Mutation of Ncf1 Ameliorates Obstruction-Induced Renal Fibrosis While Macrophage-Rescued NCF1 Further Alleviates Renal Fibrosis.

Aims: NCF1, a subunit of the NADPH oxidase 2 (NOX2), first described the expression in neutrophils and macrophages and participated in the pathogenesis from various systems. However, there are controversial findings on the role of NCF1 in different kinds of kidney diseases. In this study, we aim to pinpoint the specific role of NCF1 in the progression of renal fibrosis induced by obstruction. Results: In this study, NCF1 expression was upregulated in kidney biopsies of chronic kidney disease patients. The expression level of all subunits of the NOX2 complex was also significantly increased in the unilateral ureteral obstruction (UUO) kidney. Then, we used wild-type mice and Ncf1 mutant mice (Ncf1m1j mice) to perform UUO-induced renal fibrosis. Results demonstrated that Ncf1m1j mice exhibited mild renal fibrosis but increased macrophages count and CD11b+Ly6Chi macrophage proportion. Next, we compared the renal fibrosis degree between Ncf1m1j mice and Ncf1 macrophage-rescued mice (Ncf1m1j.Ncf1Tg-CD68 mice). We found that rescuing NCF1 expression in macrophages further alleviated renal fibrosis and decreased macrophage infiltration in the UUO kidney. In addition, flow cytometry data showed fewer CD11b+Ly6Chi macrophages in the kidney of the Ncf1m1j.Ncf1Tg-CD68 group than the Ncf1m1j group. Innovation: We first used the Ncf1m1j mice and Ncf1m1j.Ncf1Tg-CD68 mice to detect the role of NCF1 in the pathological process of renal fibrosis induced by obstruction. Also, we found that NCF1 expressed in different cell types exerts opposing effects on obstructive nephropathy. Conclusion: Taken together, our findings support that systemic mutation of Ncf1 ameliorates renal fibrosis induced by obstruction, and rescuing NCF1 in macrophages further alleviates renal fibrosis.

Decreased renal expression of PAQR5 is associated with the absence of a nephroprotective effect of progesterone in a rat UUO model

Fibrosis is a severe complication of chronic kidney disease (CKD). Progesterone, like other sex hormones, plays an important role in renal physiology, but its role in CKD is poorly understood. We investigated progesterone effect on renal fibrosis progression in the rat model of unilateral ureteral obstruction (UUO). Female rats were exposed to UUO, ovariectomy and progesterone administration after UUO with ovariectomy. Expression of key fibrosis markers, proinflammatory cytokines, levels of membrane-bound (PAQR5) and nuclear (PGR) progesterone receptors, and matrix metalloproteinase (MMP) activity were analyzed in the obstructed and intact rat kidney. In all groups exposed to UUO, decreased PAQR5 expression was observed in the obstructed kidney while in the contralateral kidney, it remained unaffected. We found increased mRNA levels for profibrotic COL1A1, FN1, MMP2, TIMP1, TIMP2, proinflammatory IL1α, IL1β, and IL18, as well as elevated α-SMA and MMP9 proteins, collagen deposition, and MMP2 activity in all UUO kidneys. Progesterone had slight or no effect on the change in these markers. Thus, we demonstrate for the first time diminished sensitivity of the kidney to progesterone associated with renal fibrosis due to a severe decrease in PAQR5 expression that was accompanied by the lack of nephroprotection in a rat UUO model.

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.

Spermidine from arginine metabolism activates Nrf2 and inhibits kidney fibrosis

Kidney metabolism may be greatly altered in chronic kidney disease. Here we report that arginine metabolism is the most altered in unilateral ureteral obstruction (UUO)-induced fibrosis of the kidneys in metabolomic analysis. Spermidine is the most increased metabolite of arginine. In human glomerulonephritis, the amount of spermidine shown by immunostaining is associated with the amount of fibrosis. In human proximal tubule cells, spermidine induces nuclear factor erythroid 2-related factor 2 (Nrf2). Subsequently, fibrotic signals, such as transforming growth factor β1 secretion, collagen 1 mRNA, and oxidative stress, represented by a decrease in the mitochondrial membrane potential is suppressed by spermidine. UUO kidneys of Arg2 knockout mice show less spermidine and significantly exacerbated fibrosis compared with wild-type mice. Nrf2 activation is reduced in Arg2 knockout UUO kidneys. Spermidine treatment prevents significant fibrotic progression in Arg2 knockout mice. Spermidine is increased in kidney fibrosis, but further increases in spermidine may reduce fibrosis.

#4161 DELETION OF PROTEIN TYROSINE KINASES 4A1 AMELIORATE RENAL FIBROSIS INDUCED BY UUO IN MICE

Abstract Background and Aims Inhibitors of protein tyrosine kinases has been investigated as potential anti-fibrotic agents. protein tyrosine kinases 4A1 belongs to a sub-class of three prenylated protein tyrosine kinases. protein tyrosine kinases 4A1 has known as promoting growth and migration of tumor cells. The role protein tyrosine kinases 4A1 has little known in kidney. We evaluated whether the protein tyrosine kinases 4A1 could be target of renal fibrosis. Method 10 weeks old male background protein tyrosine kinases 4A1 KO mice and wild type mice were divided into 4 groups; wild, protein tyrosine kinases 4A1 KO, wild with unilateral ureteral obstruction, and protein tyrosine kinases 4A1 KO with unilateral ureteral obstruction. Mice were sacrificed at 7 days after surgery and kidney tissue were collected. Molecular study and Histologic examination were performed. Results Protein tyrosine kinases 4A1 KO with unilateral ureteral obstruction mice showed decrease of renal tubule-interstitial damage and fibrosis compared to wild type unilateral ureteral obstruction mice. protein tyrosine kinases 4A1 KO with unilateral ureteral obstruction reduced the renal expression of α-SMA and TGF-β in unilateral ureteral obstruction kidney, compared to wild type with unilateral ureteral obstruction mice. Wild type with unilateral ureteral obstruction kidney showed decrease of renal expression of E-cadherin, compared to sham mice. However, protein tyrosine kinases 4A1 KO unilateral ureteral obstruction showed increase of renal expression of E-cadherin, compared to Wild type unilateral ureteral obstruction mice. In vitro, silencing of protein tyrosine kinases 4A1 in TGF-β treated HK2 cell showed increase of E-cadherin and decrease of phosphorylation of AKT and GSK3ß. Conclusion Protein tyrosine kinases 4A1 KO ameliorate renal fibrosis in unilateral ureteral obstruction kidney.

SDMA attenuates renal tubulointerstitial fibrosis through inhibition of STAT4

BackgroundRenal tubulointerstitial fibrosis is the hallmark of various chronic kidney diseases. Symmetric dimethylarginine (SDMA) is an independent cardiovascular risk factor in patients with chronic kidney diseases, which is mostly excreted through renal tubules. However, the effect of SDMA on kidneys in a pathological condition is currently unknown. In this study, we investigated the role of SDMA in renal tubulointerstitial fibrosis and explored its underlying mechanisms.MethodsMouse unilateral ureteral obstruction (UUO) and unilateral ischemia–reperfusion injury (UIRI) models were established to study renal tubulointerstitial fibrosis. SDMA was injected into kidneys through ureter retrogradely. TGF-β stimulated human renal epithelial (HK2) cells were used as an in vitro model and treated with SDMA. Signal transducer and activator of transcription-4 (STAT4) was inhibited by berbamine dihydrochloride or siRNA or overexpressed by plasmids in vitro. Masson staining and Western blotting were performed to evaluate renal fibrosis. Quantitative PCR was performed to validate findings derived from RNA sequencing analysis.ResultsWe observed that SDMA (from 0.01 to 10 µM) dose-dependently inhibited the expression of pro-fibrotic markers in TGF-β stimulated HK2 cells. Intrarenal administration of SDMA (2.5 µmol/kg or 25 µmol/kg) dose-dependently attenuated renal fibrosis in UUO kidneys. A significant increase in SDMA concentration (from 19.5 to 117.7 nmol/g, p < 0.001) in mouse kidneys was observed after renal injection which was assessed by LC–MS/MS. We further showed that intrarenal administration of SDMA attenuated renal fibrosis in UIRI induced mouse fibrotic kidneys. Through RNA sequencing analysis, we found that the expression of STAT4 was reduced by SDMA in UUO kidneys, which was further confirmed by quantitative PCR and Western blotting analysis in mouse fibrotic kidneys and renal cells. Inhibition of STAT4 by berbamine dihydrochloride (0.3 mg/ml or 3.3 mg/ml) or siRNA reduced the expression of pro-fibrotic markers in TGF-β stimulated HK2 cells. Furthermore, blockage of STAT4 attenuated the anti-fibrotic effect of SDMA in TGF-β stimulated HK2 cells. Conversely, overexpression of STAT4 reversed the anti-fibrotic effect of SDMA in TGF-β stimulated HK2 cells.ConclusionTaken together, our study indicates that renal SDMA ameliorates renal tubulointerstitial fibrosis through inhibition of STAT4.

Hederagenin ameliorates renal fibrosis in chronic kidney disease through blocking ISG15 regulated JAK/STAT signaling.

Interstitial fibrosis is the key pathological characteristics of chronic kidney diseases (CKD). In this study, we reported that hederagenin (HDG) can effectively improve the renal interstitial fibrosis and its mechanism. We constructed CKD animal models of ischemia reperfusion injury (IRI) and unilateral ureteral obstruction (UUO) respectively to observe the improvement effect of HDG on CKD. The results showed that HDG can effectively improve the pathological structure of kidney and the renal fibrosis in CKD mice. Meanwhile, HDG can also significantly reduce the expression of α-SMA and FN induced by TGF-β in Transformed C3H Mouse Kidney-1 (TCMK1) cells. Mechanistically, we performed transcriptome sequencing on UUO kidneys treated with HDG. By real time PCR screening of the sequencing results, we determined that ISG15 plays an important role in the intervention of HDG in CKD. Subsequently, we knocked-down ISG15 in TCMK1 and found that ISG15 knock-down significantly inhibited TGF-β-induced fibrotic protein expression and JAK/STAT activation. Finally, we performed electrotransfection and used liposomes to transfect ISG15 overexpression plasmids to up-regulate ISG15 in kidney and cells, respectively. We found that ISG15 can aggravate renal tubular cell fibrosis and abolish the protection of HDG on CKD. These results indicated that HDG significantly improves renal fibrosis in CKD by inhibiting ISG15 and its downstream JAK/STAT signaling pathway, which provides a new drug and research target for the subsequent treatment of CKD.

CBGA ameliorates inflammation and fibrosis in nephropathy

Cannabidiol (CBD) is thought to have multiple biological effects, including the ability to attenuate inflammatory processes. Cannabigerols (CBGA and its decarboxylated CBG molecule) have pharmacological profiles similar to CBD. The endocannabinoid system has recently emerged to contribute to kidney disease, however, the therapeutic properties of cannabinoids in kidney disease remain largely unknown. In this study, we determined whether CBD and CBGA can attenuate kidney damage in an acute kidney disease model induced by the chemotherapeutic cisplatin. In addition, we evaluated the anti-fibrosis effects of these cannabinoids in a chronic kidney disease model induced by unilateral ureteral obstruction (UUO). We find that CBGA, but not CBD, protects the kidney from cisplatin-induced nephrotoxicity. CBGA also strongly suppressed mRNA of inflammatory cytokines in cisplatin-induced nephropathy, whereas CBD treatment was only partially effective. Furthermore, both CBGA and CBD treatment significantly reduced apoptosis through inhibition of caspase-3 activity. In UUO kidneys, both CBGA and CBD strongly reduced renal fibrosis. Finally, we find that CBGA, but not CBD, has a potent inhibitory effect on the channel-kinase TRPM7. We conclude that CBGA and CBD possess reno-protective properties, with CBGA having a higher efficacy, likely due to its dual anti-inflammatory and anti-fibrotic effects paired with TRPM7 inhibition.

CircRNA_0017076 acts as a sponge for miR-185-5p in the control of epithelial-to-mesenchymal transition of tubular epithelial cells during renal interstitial fibrosis.

Renal interstitial fibrosis (RIF) is a common pathological hallmark of progressive chronic kidney disease (CKD). Circular RNAs (circRNAs) are involved in certain renal diseases, but their role in RIF is largely unknown. The present study investigated the effects and potential mechanisms of circRNA_0017076 in RIF. CircRNA_0017076 expression was markedly upregulated in transforming growth factor-β1 (TGF-β1)-treated renal tubular epithelial cells (RTECs) and kidney biopsy samples from patients with RIF. Functional assays showed that circRNA_0017076 colocalized with microRNA-185-5p (miR-185-5p) and inhibited miR-185-5p function via direct binding to miR-185-5p. In vitro, the knockdown of circRNA_0017076 inhibited the calcium ion (Ca2+) influx-mediated epithelial-to-mesenchymal transition (EMT) of RTECs and downregulated the expression of stromal interaction molecule 1 (STIM1), which is a target protein of miR-185-5p. Silencing mmu_circ_0004488 reduced fibrotic lesions in the kidneys of unilateral ureteral obstruction (UUO) mice by targeting the miR-185-5p/Stim1 axis. For the first time, we identified circRNA_0017076 as a sponge for miR-185-5p, which regulates STIM1 gene expression and is involved in RIF. Our results support circRNA_0017076 as a potential therapeutic target for RIF disease.