ELABELA-derived peptide ELA13 attenuates kidney fibrosis by inhibiting the Smad and ERK signaling pathways. / ELABELA衍生肽ELA13通过抑制Smad和ERK信号通路减轻肾纤维化.

Kidney fibrosis is an inevitable result of various chronic kidney diseases (CKDs) and significantly contributes to end-stage renal failure. Currently, there is no specific treatment available for renal fibrosis. ELA13 (amino acid sequence: RRCMPLHSRVPFP) is a conserved region of ELABELA in all vertebrates; however, its biological activity has been very little studied. In the present study, we evaluated the therapeutic effect of ELA13 on transforming growth factor-ß1 (TGF-ß1)-treated NRK-52E cells and unilateral ureteral occlusion (UUO) mice. Our results demonstrated that ELA13 could improve renal function by reducing creatinine and urea nitrogen content in serum, and reduce the expression of fibrosis biomarkers confirmed by Masson staining, immunohistochemistry, real-time polymerase chain reaction (RT-PCR), and western blot. Inflammation biomarkers were increased after UUO and decreased by administration of ELA13. Furthermore, we found that the levels of essential molecules in the mothers against decapentaplegic (Smad) and extracellular signal-regulated kinase (ERK) pathways were reduced by ELA13 treatment in vivo and in vitro. In conclusion, ELA13 protected against kidney fibrosis through inhibiting the Smad and ERK signaling pathways and could thus be a promising candidate for anti-renal fibrosis treatment.

Atg5 deficiency in macrophages protects against kidney fibrosis via the CCR6-CCL20 axis.

BACKGROUND: Autophagy is a lysosome-dependent degradation pathway that regulates macrophage activation, differentiation, and polarization. Autophagy related 5 (Atg5) is a key protein involved in phagocytic membrane elongation in autophagic vesicles that forms a complex with Atg12 and Atg16L1. Alterations in Atg5 are related to both acute and chronic kidney diseases in experimental models. However, the role of macrophage-expressed Atg5 in acute kidney injury remains unclear. METHODS: Using a myeloid cell-specific Atg5 knockout (MΦ atg5-/-) mouse, we established renal ischemia/reperfusion and unilateral ureteral obstruction models to evaluate the role of macrophage Atg5 in renal macrophage migration and fibrosis. RESULTS: Based on changes in the serum urea nitrogen and creatinine levels, Atg5 deletion had a minimal effect on renal function in the early stages after mild injury; however, MΦ atg5-/- mice had reduced renal fibrosis and reduced macrophage recruitment after 4 weeks of ischemia/reperfusion injury and 2 weeks of unilateral ureteral obstruction injury. Atg5 deficiency impaired the CCL20-CCR6 axis after severe ischemic kidneys. Chemotactic responses of bone marrow-derived monocytes (BMDMs) from MΦ atg5-/- mice to CCL20 were significantly attenuated compared with those of wild-type BMDMs, and this might be caused by the inhibition of PI3K, AKT, and ERK1/2 activation. CONCLUSIONS: Our data indicate that Atg5 deficiency decreased macrophage migration by impairing the CCL20-CCR6 axis and inhibited M2 polarization, thereby improving kidney fibrosis.

Icariin alleviates renal inflammation and tubulointerstitial fibrosis via Nrf2-mediated attenuation of mitochondrial damage.

Tubulointerstitial fibrosis is an inevitable consequence of all progressive chronic kidney disease (CKD) and contributes to a substantial health burden worldwide. Icariin, an active flavonoid glycoside obtained from Epimedium species, exerts potential antifibrotic effect. The study aimed to explore the protective effects of icariin against tubulointerstitial fibrosis in unilateral ureteral obstruction (UUO)-induced CKD mice and TGF-ß1-treated HK-2 cells, and furthermore, to elucidate the underlying mechanisms. The results demonstrated that icariin significantly improved renal function, alleviated tubular injuries, and reduced fibrotic lesions in UUO mice. Furthermore, icariin suppressed renal inflammation, reduced oxidative stress as evidenced by elevated superoxide dismutase activity and decreased malondialdehyde level. Additionally, TOMM20 immunofluorescence staining and transmission electron microscope revealed that mitochondrial mass and morphology of tubular epithelial cells in UUO mice was restored by icariin. In HK-2 cells treated with TGF-ß1, icariin markedly decreased profibrotic proteins expression, inhibited inflammatory factors, and protected mitochondria along with preserving mitochondrial morphology, reducing reactive oxygen species (ROS) and mitochondrial ROS (mtROS) overproduction, and preserving membrane potential. Further investigations demonstrated that icariin could activate nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) pathway both in vivo and in vitro, whereas inhibition of Nrf2 by ML385 counteracted the protective effects of icariin on TGF-ß1-induced HK-2 cells. In conclusion, icariin protects against renal inflammation and tubulointerstitial fibrosis at least partly through Nrf2-mediated attenuation of mitochondrial dysfunction, which suggests that icariin could be developed as a promising therapeutic candidate for the treatment of CKD.

LncRNA Meg3 Aggravates Renal Fibrosis Caused by Unilateral Ureteral Obstruction in Rats by Activating the Hedgehog Pathway.

BACKGROUND: The hedgehog signaling pathway exerts vital functions in regulating epithelial-to-mesenchymal transition (EMT) in renal interstitial fibrosis (RIF). It was reported that lncRNA-maternally expressed gene 3 (lncRNA Meg3) can regulate hepatic fibrosis by regulating the expression of smoothened (Smo) in the hedgehog signaling pathway. However, the specific role of lncRNA Meg3 in renal fibrosis resulting from unilateral ureteral obstruction (UUO) by regulating the hedgehog signaling pathway has not been reported. Hence, this research aimed to expound the effects of lncRNA Meg3 on renal fibrosis induced by UUO in rats via the hedgehog pathway. METHODS: Peripheral blood was collected from patients with chronic kidney disease (CKD, CKD group) and healthy volunteers (Normal group) at the same period. In addition, 6-week-old male Sprague-Dawley (SD) rats were divided to Sham, UUO, UUO+shRNA Negative control (shNC), and UUO+sh-Meg3 groups, and their kidney tissues and serum were gathered. Next, quantitative real-time polymerase chain reaction (qRT-PCR) was employed for detecting the lncRNA Meg3 expression level in the serum of patients and renal tissue of rats; kits for testing levels of blood urea nitrogen (BUN), creatinine (Cr), hydroxyproline (HYP), and 24-hour urine protein (24-up) in rats of each group; hematoxylin and eosin (HE) staining and Masson staining for observing kidney tissue and renal fibrosis level in rats; western blot for measuring levels of collagen type III (Col III), α-Smooth muscle actin (α-SMA), fibronectin, E-cadherin, sonic hedgehog (Shh), patched (Ptch) protein, smoothened (Smo) protein and glioma-associated oncogene homolog 1 (Gli1) protein expression. RESULTS: LncRNA Meg3 was highly expressed in CKD patients and UUO rats (p < 0.01). In contrast to the UUO+shNC group, knocking down lncRNA Meg3 improved renal injury, relieved pathological renal lesions, and reduced kidney fibrosis and related protein levels. It inhibited the hedgehog pathway in kidney tissues of UUO rats (p < 0.05 and p < 0.01). CONCLUSIONS: LncRNA Meg3 can aggravate UUO-induced rat renal fibrosis by activating the hedgehog pathway.

Integrative transcriptomic and proteomic profile revealed inhibition of oxidative phosphorylation and peroxisomes during renal interstitial fibrosis.

Effective therapies of chronic kidney disease (CKD) are lacking due to the unclear molecular pathogenesis. Previous single omics-studies have described potential molecular regulation mechanism of CKD only at the level of transcription or translation. Therefore, this study generated an integrated transcriptomic and proteomic profile to provide deep insights into the continuous transcription-translation process during CKD. The comprehensive datasets identified 14,948 transcripts and 6423 proteins, 233 up-regulated and 364 down-regulated common differentially expressed genes of transcriptome and proteome were selected to further combined bioinformatics analysis. The obtained results revealed reactive oxygen species (ROS) metabolism and antioxidant system due to imbalance of mitochondria and peroxisomes were significantly repressed in CKD. Overall, this study presents a valuable multi-omics analysis that sheds light on the molecular mechanisms underlying CKD. SIGNIFICANCE: Chronic kidney disease (CKD) is a progressive and irreversible condition that results in abnormal kidney function and structure, and is ranked 18th among the leading causes of death globally, leading to a significant societal burden. Hence, there is an urgent need for research to detect new, sensitive, and specific biomarkers. Omics-based studies offer great potential to identify underlying disease mechanisms, aid in clinical diagnosis, and develop novel treatment strategies for CKD. Previous studies have mainly focused on the regulation of gene expression or protein synthesis in CKD, thereby compelling us to conduct a meticulous analysis of transcriptomic and proteomic data from the UUO mouse model. Here, we have performed a unified analysis of CKD model by integrating transcriptomes and protein suites for the first time. Our study contributes to a deeper understanding of the pathogenesis of CKD and provides a basis for subsequent disease management and drug development.

Demethylzeylasteral protects against renal interstitial fibrosis by attenuating mitochondrial complex I-mediated oxidative stress.

ETHNOPHARMACOLOGICAL RELEVANCE: Renal interstitial fibrosis (RIF) is a main pathological process in chronic kidney disease (CKD). Demethylzeylasteral (DML), a major component of Tripterygium wilfordii Hook. f., has anti-renal fibrosis effects. However, its mechanism of action remains incompletely understood. AIM OF THE STUDY: The present study was designed to comprehensively examine the effects of DML on RIF and the underlying mechanisms. MATERIALS AND METHODS: Pathological experiments were performed to determine the therapeutic effect of DML on a mouse model of UUO-induced RIF. To determine the novel mechanisms underlying the therapeutic effects of DML against RIF, a comprehensive transcriptomics analysis was performed on renal tissues, which was further verified by a series of experiments. RESULTS: Pathological and immunohistochemical staining showed that DML inhibited UUO-induced renal damage and reduced the expression of fibrosis-related proteins in mice. Transcriptomic analysis revealed that the partial subunits of mitochondrial complex (MC) I and II may be targets by which DML protects against RIF. Furthermore, DML treatment reduced mitochondrial reactive oxygen species (ROS) levels, consequently promoting ATP production and mitigating oxidative stress-induced injury in mice and cells. Notably, this protective effect was attributed to the inhibition of MC I activity, suggesting a crucial role for this specific complex in mediating the therapeutic effects of DML against RIF. CONCLUSIONS: This study provides compelling evidence that DML may be used to treat RIF by effectively suppressing mitochondrial oxidative stress injury mediated by MC I. These findings offer valuable insights into the pharmacological mechanisms of DML and its potential clinical application for patients with CKD.

Beclin-1-Derived Peptide MP1 Attenuates Renal Fibrosis by Inhibiting the Wnt/ß-Catenin Pathway.

Renal fibrosis is distinguished by the abnormal deposition of extracellular matrix and progressive loss of nephron function, with a lack of effective treatment options in clinical practice. In this study, we discovered that the Beclin-1-derived peptide MP1 significantly inhibits the abnormal expression of fibrosis and epithelial-mesenchymal transition-related markers, including α-smooth muscle actin, fibronectin, collagen I, matrix metallopeptidase 2, Snail1, and vimentin both in vitro and in vivo. H&E staining was employed to evaluate renal function, while serum creatinine (Scr) and blood urea nitrogen (BUN) were used as main indices to assess pathologic changes in the obstructed kidney. The results demonstrated that daily treatment with MP1 during the 14-day experiment significantly alleviated renal dysfunction and changes in Scr and BUN in mice with unilateral ureteral obstruction. Mechanistic research revealed that MP1 was found to have a significant inhibitory effect on the expression of crucial components involved in both the Wnt/ß-catenin and transforming growth factor (TGF)-ß/Smad pathways, including ß-catenin, C-Myc, cyclin D1, TGF-ß1, and p-Smad/Smad. However, MP1 exhibited no significant impact on either the LC3II/LC3I ratio or P62 levels. These findings indicate that MP1 improves renal physiologic function and mitigates the fibrosis progression by inhibiting the Wnt/ß-catenin pathway. Our study suggests that MP1 represents a promising and novel candidate drug precursor for the treatment of renal fibrosis. SIGNIFICANCE STATEMENT: This study indicated that the Beclin-1-derived peptide MP1 effectively mitigated renal fibrosis induced by unilateral ureteral obstruction through inhibiting the Wnt/ß-catenin pathway and transforming growth factor-ß/Smad pathway, thereby improving renal physiological function. Importantly, unlike other Beclin-1-derived peptides, MP1 exhibited no significant impact on autophagy in normal cells. MP1 represents a promising and novel candidate drug precursor for the treatment of renal fibrosis focusing on Beclin-1 derivatives and Wnt/ß-catenin pathway.

Kangxianling formula attenuates renal fibrosis by regulating gut microbiota.

BACKGROUND: Renal fibrosis (RF) produced adverse effect on kidney function. Recently, intestinal dysbiosis is a key regulator that promotes the formation of renal fibrosis. This study will focus on exploring the protective mechanism of Kangxianling Formula (KXL) on renal fibrosis from the perspective of intestinal flora. METHODS: Unilateral Ureteral Obstruction (UUO) was used to construct rats' model with RF, and receive KXL formula intervention for 1 week. The renal function indicators were measured. Hematoxylin-eosin (HE), Masson and Sirus red staining were employed to detect the pathological changes of renal tissue in each group. The expression of α-SMA, Col-III, TGF-ß, FN, ZO-1, and Occuludin was detected by immunofluorescence and immunohistochemistry. Rat feces samples were collected and analyzed for species' diversity using high-throughput sequencing 16S rRNA. RESULTS: Rats in UUO groups displayed poor renal function as well as severe RF. The pro-fibrotic protein expression in renal tissues including α-SMA, Col-III, TGF-ß and FN was increased in UUO rats, while ZO-1 and Occuludin -1 expression was downregulated in colon tissues. The above changes were attenuated by KXL treatment. 16S rRNA sequencing results revealed that compared with the sham group, the increased abundance of pathogenic bacteria including Acinetobacter, Enterobacter and Proteobacteria and the decreased abundance of beneficial bacteria including Actinobacteriota, Bifidobacteriales, Prevotellaceae, and Lactobacillus were found in UUO group. After the administration of KXL, the growth of potential pathogenic bacteria was reduced and the abundance of beneficial bacteria was enhanced. CONCLUSION: KXL displays a therapeutical potential in protecting renal function and inhibiting RF, and its mechanism of action may be associated with regulating intestinal microbiota.

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 in vivo model and treated tubular epithelial cells (TECs) with transforming growth factor (TGF)-ß1 as in vitro models. Our findings revealed increased FTO expression in UUO mouse model and TGF-ß1-treated TECs. By modulating FTO expression through FTO heterozygous mutation mice (FTO+/- ) in vivo and small interfering RNA (siRNA) in vitro, we observed attenuation of UUO and TGF-ß1-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-ß1-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-ß1-induced RUNX1 elevation in vivo and in vitro. 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.

Impaired TFEB-mediated autophagy-lysosome fusion promotes tubular cell cycle G2/M arrest and renal fibrosis by suppressing ATP6V0C expression and interacting with SNAREs.

Increasing evidence suggests that autophagy plays a major role during renal fibrosis. Transcription factor EB (TFEB) is a critical regulator of autophagy- and lysosome-related gene transcription. However, the pathophysiological roles of TFEB in renal fibrosis and fine-tuned mechanisms by which TFEB regulates fibrosis remain largely unknown. Here, we found that TFEB was downregulated in unilateral ureteral obstruction (UUO)-induced human and mouse fibrotic kidneys, and kidney-specific TFEB overexpression using recombinant AAV serotype 9 (rAAV9)-TFEB in UUO mice alleviated renal fibrosis pathogenesis. Mechanically, we found that TFEB's prevention of extracellular matrix (ECM) deposition depended on autophagic flux integrity and its subsequent blockade of G2/M arrest in tubular cells, rather than the autophagosome synthesis. In addition, we together RNA-seq with CUT&Tag analysis to determine the TFEB targeted gene ATP6V0C, and revealed that TFEB was directly bound to the ATP6V0C promoter only at specific site to promote its expression through CUT&Run-qPCR and luciferase reporter assay. Interestingly, TFEB induced autophagic flux integrity, mainly dependent on scaffold protein ATP6V0C-mediated autophagosome-lysosome fusion by bridging with STX17 and VAMP8 (major SNARE complex) by co-immunoprecipitation analysis, rather than its mediated lysosomal acidification and degradation function. Moreover, we further investigated the underlying mechanism behind the low expression of TEFB in UUO-induced renal fibrosis, and clearly revealed that TFEB suppression in fibrotic kidney was due to DNMT3a-associated TFEB promoter hypermethylation by utilizing methylation specific PCR (MSP) and bisulfite-sequencing PCR (BSP), which could be effectively recovered by 5-Aza-2'-deoxycytidine (5A-za) to alleviate renal fibrosis pathogenesis. These findings reveal for the first time that impaired TFEB-mediated autophagosome-lysosome fusion disorder, tubular cell G2/M arrest and renal fibrosis appear to be sequentially linked in UUO-induced renal fibrosis and suggest that DNMT3a/TFEB/ATP6V0C may serve as potential therapeutic targets to prevent renal fibrosis.

Herb pair of Rhubarb-Astragalus mitigates renal interstitial fibrosis through downregulation of autophagy via p38-MAPK/TGF-ß1 and p38-MAPK/smad2/3 pathways.

BACKGROUND: Chronic kidney disease (CKD) has a high incidence and poor prognosis; however, no effective treatment is currently available. Our previous study found that the improvement effect of the herb pair of Rhubarb-Astragalus on CKD is likely related to the inhibition of the TGF-ß1/p38-MAPK pathway. In the present study, a p38-MAPK inhibitor was used to further investigate the inhibitory effect of Rhubarb-Astragalus on the TGF-ß1/p38-MAPK pathway and its relationship with autophagy. METHODS: A rat model of unilateral ureteral obstruction (UUO) was established, and a subgroup of rats was administered Rhubarb-Astragalus. Renal function and renal interstitial fibrosis (RIF) were assessed 21 d after UUO induction. In vitro, HK-2 cells were treated with TGF-ß1 and a subset of cells were treated with Rhubarb-Astragalus or p38-MAPK inhibitor. Western blotting, immunohistochemistry, and qRT-PCR analyses were used to detect the relevant protein and mRNA levels. Transmission electron microscopy was used to observe autophagosomes. RESULTS: Rhubarb-Astragalus treatment markedly decreased the elevated levels of blood urea nitrogen, serum creatinine, and urinary N-acetyl-ß-D-glucosaminidase; attenuated renal damage and RIF induced by UUO; and reduced the number of autophagosomes and lysosomes in UUO-induced renal tissues. Additionally, Rhubarb-Astragalus reduced the protein and mRNA levels of α-SMA, collagen I, LC3, Atg3, TGF-ß1, p38-MAPK, smad2/3, and TAK1 in renal tissues of UUO rats. Rhubarb-Astragalus also reduced protein and mRNA levels of these indicators in vitro. Importantly, the effect of the p38-MAPK inhibitor was similar to that of Rhubarb-Astragalus. CONCLUSIONS: Rhubarb-Astragalus improves CKD possibly by downregulating autophagy via the p38-MAPK/TGF-ß1 and p38-MAPK/smad2/3 pathways.

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

BACKGROUND: Chronic 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. METHODS: We 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. FINDINGS: Cebpa 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-ß. 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. INTERPRETATION: Our 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.

Kidney Injury by Unilateral Ureteral Obstruction in Mice Lacks Sex Differences.

INTRODUCTION: Renal fibrosis is a critical event in the development and progression of chronic kidney disease (CKD), and it is considered the final common pathway for all types of CKD. The prevalence of CKD is higher in females; however, males have a greater prevalence of end-stage renal disease. In addition, low birth weight and low nephron number are associated with increased risk for CKD. This study examined the development and severity of unilateral ureter obstruction (UUO)-induced renal fibrosis in male and female wild-type (ROP +/+) and mutant (ROP Os/+) mice, a mouse model of low nephron number. METHODS: Male and female ROP +/+ and ROP Os/+ mice were subjected to UUO, and kidney tissue was collected at the end of the 10-day experimental period. Kidney histological analysis and mRNA expression determined renal fibrosis, tubular injury, collagen deposition, extracellular matrix proteins, and immune cell infiltration. RESULTS: Male and female UUO mice demonstrated marked renal injury, kidney fibrosis, and renal extracellular matrix production. Renal fibrosis and α-smooth muscle actin were increased to a similar degree in ROP +/+ and ROP Os/+ mice with UUO of either sex. There were also no sex differences in renal tubular cast formation or renal infiltration of macrophage in ROP +/+ and ROP Os/+ UUO mice. Interestingly, renal fibrosis and α-smooth muscle actin were 1.5-3-fold greater in UUO-ROP +/+ compared to UUO-ROP Os/+ mice. Renal inflammation phenotypes following UUO were also 30-45% greater in ROP +/+ compared to ROP Os/+ mice. Likewise, expression of extracellular matrix and renal fibrotic genes was greater in UUO-ROP +/+ mice compared to UUO-ROP Os/+ mice. In contrast to these findings, ROP Os/+ mice with UUO demonstrated glomerular hypertrophy with 50% greater glomerular tuft area compared to ROP +/+ with UUO. Glomerular hypertrophy was not sex-dependent in any of the genotypes of ROP mice. These findings provide evidence that low nephron number contributes to UUO-induced glomerular hypertrophy in ROP Os/+ mice but does not enhance renal fibrosis, inflammation, and renal tubular injury. CONCLUSION: Taken together, we demonstrate that low nephron number contributes to enhanced glomerular hypertrophy but not kidney fibrosis and tubular injury. We also demonstrate that none of the changes caused by UUO was affected by sex in any of the ROP mice genotypes.

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

BACKGRUOUND: 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. METHODS: 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-ß-treated renal cells in vitro. RESULTS: 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-ßstimulated expression of fibronectin, collagen type I, plasminogen activator inhibitor-1 (PAI-1), and α-SMA. In HK-2 cells, alantolactone inhibited TGF-ß-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-ß secretion but also Smad3 phosphorylation and translocation to nucleus in both kidney cell lines. CONCLUSION: Alantolactone improves renal fibrosis by inhibiting the TGF-ß/Smad3 signaling pathway in obstructive nephropathy. Thus, alantolactone is a potential therapeutic agent for chronic kidney disease.

The progression of obstructive renal fibrosis in rats is regulated by ADAMTS18 gene methylation in the embryonic kidney through the AKT/Notch pathway.

This study aimed to explore the mechanism by which postembryonic renal ADAMTS18 methylation influences obstructive renal fibrosis in rats. After exposure to transforming growth factor (TGF)-ß1 during the embryonic period, analysis of postembryonic renal ADAMTS18 methylation and expression levels was conducted. Histological analysis was performed to assess embryonic kidney lesions and damage. Western blot analysis was used to determine the expression of renal fibrosis markers. Rats with ureteral obstruction and a healthy control group were selected. The methylation levels of ADAMTS18 in the different groups were analyzed. Western blot analysis and immunohistochemistry were performed to analyze the expression of renal fibrosis markers, and kidney-related indicators were measured. Treatment with TGF-ß1 resulted in abnormal development of the postembryonic kidney, which was characterized by rough kidney surfaces with mild depressions and irregularities on the outer surface. TGF-ß1 treatment significantly promoted ADAMTS18 methylation and activated the protein kinase B (AKT)/Notch pathway. Ureteral obstruction was induced to establish a renal hydronephrosis model, which led to renal fibrotic injury in newborn rats. Overexpression of the ADAMTS18 gene alleviated renal fibrosis. The western blot results showed that compared to that in the control group, the expression of renal fibrosis markers was significantly decreased after ADAMTS18 overexpression, and there was a thicker renal parenchymal tissue layer and significantly reduced p-AKT/AKT and Notch1 levels. TGF-ß1 can induce ADAMTS18 gene methylation in the postembryonic kidney, and the resulting downregulation of ADAMTS18 expression has long-term effects on kidney development, potentially leading to increased susceptibility to obstructive renal fibrosis. This mechanism may involve activation of the AKT/Notch pathway. Reversing ADAMTS18 gene methylation may reverse this process.

HIF-1 contributes to autophagy activation via BNIP3 to facilitate renal fibrosis in hypoxia in vitro and UUO in vivo.

The molecular basis of renal interstitial fibrosis, a major pathological feature of progressive kidney diseases, remains poorly understood. Autophagy has been implicated in renal fibrosis, but whether it promotes or inhibits fibrosis remains controversial. Moreover, it is unclear how autophagy is activated and sustained in renal fibrosis. The present study was designed to address these questions using the in vivo mouse model of unilateral ureteral obstruction and the in vitro model of hypoxia in renal tubular cells. Both models showed the activation of hypoxia-inducible factor-1 (HIF-1) and autophagy along with fibrotic changes. Inhibition of autophagy with chloroquine reduced renal fibrosis in unilateral ureteral obstruction model, whereas chloroquine and autophagy-related gene 7 knockdown decreased fibrotic changes in cultured renal proximal tubular cells, supporting a profibrotic role of autophagy. Notably, pharmacological and genetic inhibition of HIF-1 led to the suppression of autophagy and renal fibrosis in these models. Mechanistically, knock down of BCL2 and adenovirus E1B 19-kDa-interacting protein 3 (BNIP3), a downstream target gene of HIF, decreased autophagy and fibrotic changes during hypoxia in BUMPT cells. Together, these results suggest that HIF-1 may activate autophagy via BNIP3 in renal tubular cells to facilitate the development of renal interstitial fibrosis.NEW & NOTEWORTHY Autophagy has been reported to participate in renal fibrosis, but its role and underlying activation mechanism is unclear. In this study, we report the role of HIF-1 in autophagy activation in models of renal fibrosis and further investigate the underlying mechanism.

Fibroblast growth factor 21 alleviates unilateral ureteral obstruction-induced renal fibrosis by inhibiting Wnt/ß-catenin signaling pathway.

Fibroblast growth factor 21 (FGF21) is a key regulator of energy metabolism. Recent studies suggested that serum FGF21 levels increase with declining renal function. However, the link between FGF21 and kidney diseases and the direct effect of FGF21 in renal fibrosis remains unclear. In this study, FGF21 was upregulated in unilateral ureteral obstruction (UUO)-induced renal fibrosis and cellular fibrosis induced by transforming growth factor-ß, and renal expression of FGF21 was positively correlated with fibrosis markers. Additionally, FGF21 was regulated by Wnt/ß-catenin signaling pathway. The knockdown and overexpression of FGF21 in mouse tubular epithelial cells demonstrated that FGF21 alleviates renal fibrosis by inhibiting the Wnt/ß-catenin signaling pathway. To investigate the effect of FGF21 on renal fibrosis in vivo, we established an overexpression model by injecting the plasmid in mice and found that FGF21 overexpression relieved UUO-induced renal fibrosis and renal inflammatory response. Taken together, FGF21 is upregulated with the activation of Wnt/ß-catenin signaling pathway and alleviates renal fibrosis by inhibiting the activation of Wnt/ß-catenin signaling pathway in a negative feedback mode. These results provide a new understanding for the source of elevated serum FGF21 in patients with chronic kidney disease and prove that FGF21 is a direct inhibitor of the progression of renal fibrosis, thus providing novel therapeutic intervention insights for renal fibrosis.

Fisetin ameliorates fibrotic kidney disease in mice via inhibiting ACSL4-mediated tubular ferroptosis.

Kidney fibrosis is the hallmark of chronic kidney disease (CKD) progression, whereas no effective anti-fibrotic therapies exist. Recent evidence has shown that tubular ferroptosis contributes to the pathogenesis of CKD with persistent proinflammatory and profibrotic responses. We previously reported that natural flavonol fisetin alleviated septic acute kidney injury and protected against hyperuricemic nephropathy in mice. In this study, we investigated the therapeutic effects of fisetin against fibrotic kidney disease and the underlying mechanisms. We established adenine diet-induced and unilateral ureteral obstruction (UUO)-induced CKD models in adult male mice. The two types of mice were administered fisetin (50 or 100 mg·kg-1·d-1, i.g.) for 3 weeks or 7 days, respectively. At the end of the experiments, the mice were euthanized, and blood and kidneys were gathered for analyzes. We showed that fisetin administration significantly ameliorated tubular injury, inflammation, and tubulointerstitial fibrosis in the two types of CKD mice. In mouse renal tubular epithelial (TCMK-1) cells, treatment with fisetin (20 µM) significantly suppressed adenine- or TGF-ß1-induced inflammatory responses and fibrogenesis, and improved cell viability. By quantitative real-time PCR analysis of ferroptosis-related genes, we demonstrated that fisetin treatment inhibited ferroptosis in the kidneys of CKD mice as well as in injured TCMK-1 cells, as evidenced by decreased ACSL4, COX2, and HMGB1, and increased GPX4. Fisetin treatment effectively restored ultrastructural abnormalities of mitochondrial morphology and restored the elevated iron, the reduced GSH and GSH/GSSG as well as the increased lipid peroxide MDA in the kidneys of CKD mice. Notably, abnormally high expression of the ferroptosis key marker ACSL4 was verified in the renal tubules of CKD patients (IgAN, MN, FSGS, LN, and DN) as well as adenine- or UUO-induced CKD mice, and in injured TCMK-1 cells. In adenine- and TGF-ß1-treated TCMK-1 cells, ACSL4 knockdown inhibited tubular ferroptosis, while ACSL4 overexpression blocked the anti-ferroptotic effect of fisetin and reversed the cytoprotective, anti-inflammatory, and anti-fibrotic effects of fisetin. In summary, we reveal a novel aspect of the nephroprotective effect of fisetin, i.e. inhibiting ACSL4-mediated tubular ferroptosis against fibrotic kidney diseases.

SNHG16/miR-205/HDAC5 is involved in the progression of renal fibrosis.

Renal interstitial fibrosis (RIF) represents an irreversible and progressive pathological manifestation of chronic renal disease, which ultimately leads to end-stage renal disease. Long noncoding RNAs (lncRNAs) have been suggested to be involved in the progression of RIF. Small nucleolar RNA host gene 16 (SNHG16), a member of lncRNAs, has been found to be involved in the progression of pulmonary fibrosis. This paper first researched the effect of SNHG16 on renal fibrosis. We established a unilateral ureteral obstruction (UUO)-induced mouse RIF model by ligation of the left ureter to evaluate the biological function of SNHG16 in RIF. As a result, SNHG16 was upregulated in UUO-induced renal fibrotic tissues. Knockdown of SNHG16 inhibited RIF and reduced alpha-smooth muscle actin (α-SMA), fibronectin, and college IV expression. miR-205 was a target of SNHG16, and downregulated in UUO-induced renal fibrotic tissues. Inhibition of miR-205 promoted RIF and increased the expression of α-SMA, college IV, and fibronectin. Overexpression of SNHG16 promoted the UUO-induced RIF, but miR-205 abrogated this effect of SNHG16. Histone deacetylase 5 (HDAC5) showed high expression in UUO-induced renal fibrotic tissues. Knockdown of HDAC5 significantly reduced α-SMA, fibronectin, and college IV expression in renal tissues of UUO-induced mice. Inhibition of miR-205 promoted HDAC5 expression, but knockdown of SNHG16 inhibited HDAC5 expression in renal tissues of UUO-induced mice. In conclusion, SHNG16 is highly expressed in renal fibrotic tissues of UUO-induced mice. Knockdown of SHNG16 may prevent UUO-induced RIF by indirectly upregulating HDAC5 via targeting miR-205. SHNG16 may be novel target for treating renal fibrosis.

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.