Fibrotic Kidney Disease Research Articles

Efficient and selective kidney targeting by chemically modified carbohydrate conjugates

We investigated a renal tubule-targeting carbohydrate (RENTAC) that can selectively deliver small-molecule and nucleic acid analogs to the proximal convoluted tubules of the kidney following systemic delivery in mice. We comprehensively evaluated anti-miR-21-peptide nucleic acid-RENTAC, and fluorophore-RENTAC conjugates in cell culture and invivo. We established that RENTAC conjugates showed megalin- and cubilin-dependent endocytic uptake in the immortalized kidney cell line. Invivo biodistribution studies confirmed the retention of RENTAC conjugates in the kidneys for several days compared with other organs. Immunofluorescence staining confirmed the selective distribution of the RENTAC conjugates in proximal convoluted tubules. We further demonstrated proximal convoluted tubule targeting features of RENTAC conjugates in a folic acid-induced kidney fibrosis mouse model. As a biological readout, we targeted miR-33 using antisense peptide nucleic acid (PNA) 33-RENTAC conjugates in the fibrotic kidney disease model. The targeted delivery of PNA 33-RENTAC resulted in slower fibrosis progression and decreased collagen deposition. We also confirmed that the RENTAC ligand did not exert any adverse reactions. Thus, we established that the RENTAC ligand can be used for broad clinical applications targeting the kidneys selectively.

#2394 Inhibition of ACSS2-mediated H3K9 crotonylation alleviates kidney fibrosis via IL-1β-dependent macrophage activation and tubular cell senescence

Abstract Background and Aims Histone lysine crotonylation (Kcr), a novel posttranslational modification, is widespread as acetylation (Kac); however, its roles are largely unknown in kidney fibrosis. Method Human renal tissue, fixed in formaldehyde, and embedded in paraffin, was selected from the files of the Service of Pathology of West China Hospital, Sichuan University.ACSS2 knockout mice and tubular specific knock out of ACSS2 mice in C57BL/6N background and wild-type littermates (WT) were used to induce the FAN and UUO model or as control groups (n = 6 each). In the parallel study, C57BL/6N mice were randomly divided into three groups: control, FAN/UUO and FAN/UUO+ACSS2 inhibitor (n = 6 each). At day 7, mice were sacrificed and the serum creatinine, BUN and urinary albumin/creatinine ratio (ACR) were tested. Renal histological injury was measured by HE and Masson's trichrome staining. SA-β-gal staining was used to check cellular senescence. Renal tissues of the mice were analyzed by western blot and qPCR assay for the expression of ACSS2, H3K9cr, fibrosis and cellular senescence related proteins and genes. RNA-sequencing and ChIP-sequencing combined with in vitro cell experiments were used to figure out the underlying mechanism. Results In this study, we reported that histone Kcr of tubular epithelial cells was abnormally elevated in fibrotic kidneys. By screening these crotonylated/acetylated factors, a crotonyl-CoA-producing enzyme ACSS2 (acyl-CoA synthetase short chain family member 2) was found to remarkably increase histone 3 lysine 9 crotonylation (H3K9cr) level without influencing H3K9ac in kidneys and tubular epithelial cells. The integrated analysis of ChIP-seq and RNA-seq of fibrotic kidneys revealed that the hub proinflammatory cytokine IL-1β, which is regulated by H3K9cr, play crucial roles in fibrogenesis. Furthermore, genetic and pharmacologic inhibition of ACSS2 both suppressed H3K9cr-mediated IL-1β expression, which thereby alleviated IL-1β-dependent macrophage activation and tubular cell senescence to delay renal fibrosis. Conclusion Collectively, our findings uncover that H3K9cr exerts a critical, previously unrecognized role in kidney fibrosis, where ACSS2 represents an attractive drug target to slow fibrotic kidney disease progression.

Inhibition of ACSS2-mediated histone crotonylation alleviates kidney fibrosis via IL-1β-dependent macrophage activation and tubular cell senescence

Histone lysine crotonylation (Kcr), as a posttranslational modification, is widespread as acetylation (Kac); however, its roles are largely unknown in kidney fibrosis. In this study, we report that histone Kcr of tubular epithelial cells is abnormally elevated in fibrotic kidneys. By screening these crotonylated/acetylated factors, a crotonyl-CoA-producing enzyme ACSS2 (acyl-CoA synthetase short chain family member 2) is found to remarkably increase histone 3 lysine 9 crotonylation (H3K9cr) level without influencing H3K9ac in kidneys and tubular epithelial cells. The integrated analysis of ChIP-seq and RNA-seq of fibrotic kidneys reveal that the hub proinflammatory cytokine IL-1β, which is regulated by H3K9cr, play crucial roles in fibrogenesis. Furthermore, genetic and pharmacologic inhibition of ACSS2 both suppress H3K9cr-mediated IL-1β expression, which thereby alleviate IL-1β-dependent macrophage activation and tubular cell senescence to delay renal fibrosis. Collectively, our findings uncover that H3K9cr exerts a critical, previously unrecognized role in kidney fibrosis, where ACSS2 represents an attractive drug target to slow fibrotic kidney disease progression.

Identification of common and specific fibrosis-related genes in three common chronic kidney diseases

Background Kidney fibrosis is the common final pathway of virtually all advanced forms of chronic kidney disease (CKD) including diabetic nephropathy (DN), IgA nephropathy (IgAN) and membranous nephropathy (MN), with complex mechanism. Comparative gene expression analysis among these types of CKD may shed light on its pathogenesis. Therefore, we conducted this study aiming at exploring the common and specific fibrosis-related genes involved in different types of CKD. Methods Kidney biopsy specimens from patients with different types of CKD and normal control subjects were analyzed using the NanoString nCounter® Human Fibrosis V2 Panel. Genes differentially expressed in all fibrotic DN, IgAN and MN tissues compared to the normal controls were regarded as the common fibrosis-related genes in CKD, whereas genes exclusively differentially expressed in fibrotic DN, IgAN or MN samples were considered to be the specific genes related to fibrosis in DN, IgAN and MN respectively. Quantitative real-time PCR (qRT-PCR) was performed to validate the expression of the selected genes. Results Protein tyrosine phosphatase receptor type C (PTPRC), intercellular cell adhesion molecule-1 (ICAM1), vascular cell adhesion molecule-1 (VCAM1), interleukin 10 receptor alpha (IL10RA) and CC chemokine receptor 2 (CCR2) were identified as the potential common genes for kidney fibrosis in different types of CKD, while peroxisome proliferator-activated receptor alpha (PPARA), lactate oxidase (LOX), secreted phosphoprotein 1 (SPP1) were identified as the specific fibrosis-associated genes for DN, IgAN and MN respectively. qRT-PCR demonstrated that the expression levels of these selected genes were consistent with the NanoString analysis. Conclusions There were both commonalities and differences in the mechanisms of fibrosis in different types of CKD, the commonalities might be used as the common therapeutic targets for kidney fibrosis in CKD, while the differences might be used as the diagnostic markers for DN, IgAN and MN respectively. Inflammation was highly relevant to the pathogenesis of fibrosis. This study provides further insight into the pathophysiology and treatment of fibrotic kidney disease.

Single-cell sequencing dissects the transcriptional identity of activated fibroblasts and identifies novel persistent distal tubular injury patterns in kidney fibrosis

Examining kidney fibrosis is crucial for mechanistic understanding and developing targeted strategies against chronic kidney disease (CKD). Persistent fibroblast activation and tubular epithelial cell (TEC) injury are key CKD contributors. However, cellular and transcriptional landscapes of CKD and specific activated kidney fibroblast clusters remain elusive. Here, we analyzed single cell transcriptomic profiles of two clinically relevant kidney fibrosis models which induced robust kidney parenchymal remodeling. We dissected the molecular and cellular landscapes of kidney stroma and newly identified three distinctive fibroblast clusters with “secretory”, “contractile” and “vascular” transcriptional enrichments. Also, both injuries generated failed repair TECs (frTECs) characterized by decline of mature epithelial markers and elevation of stromal and injury markers. Notably, frTECs shared transcriptional identity with distal nephron segments of the embryonic kidney. Moreover, we identified that both models exhibited robust and previously unrecognized distal spatial pattern of TEC injury, outlined by persistent elevation of renal TEC injury markers including Krt8 and Vcam1, while the surviving proximal tubules (PTs) showed restored transcriptional signature. We also found that long-term kidney injuries activated a prominent nephrogenic signature, including Sox4 and Hox gene elevation, which prevailed in the distal tubular segments. Our findings might advance understanding of and targeted intervention in fibrotic kidney disease.

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-β1-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-β1-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.

Inhibition of ACSL4 ameliorates tubular ferroptotic cell death and protects against fibrotic kidney disease

Ferroptosis is a recently recognized form of regulated cell death, characterized by iron-dependent accumulation of lipid peroxidation. Ample evidence has depicted that ferroptosis plays an essential role in the cause or consequence of human diseases, including cancer, neurodegenerative disease and acute kidney injury. However, the exact role and underlying mechanism of ferroptosis in fibrotic kidney remain unknown. Acyl-CoA synthetase long-chain family member 4 (ACSL4) has been demonstrated as an essential component in ferroptosis execution by shaping lipid composition. In this study, we aim to discuss the potential role and underlying mechanism of ACSL4-mediated ferroptosis of tubular epithelial cells (TECs) during renal fibrosis. The unbiased gene expression studies showed that ACSL4 expression was tightly associated with decreased renal function and the progression of renal fibrosis. To explore the role of ACSL4 in fibrotic kidney, ACSL4 specific inhibitor rosiglitazone (ROSI) was used to disturb the high expression of ACSL4 in TECs induced by TGF-β, unilateral ureteral obstruction (UUO) and fatty acid (FA)-modeled mice in vivo, and ACSL4 siRNA was used to knockdown ACSL4 in TGF-β-induced HK2 cells in vitro. The results demonstrated that inhibition and knockdown of ACSL4 effectively attenuated the occurrence of ferroptosis in TECs and alleviated the interstitial fibrotic response. In addition, the expression of various profibrotic cytokines all decreased after ROSI-treated in vivo and in vitro. Further investigation showed that inhibition of ACSL4 obviously attenuates the progression of renal fibrosis by reducing the proferroptotic precursors arachidonic acid- and adrenic acid- containing phosphatidylethanolamine (AA-PE and AdA-PE). In conclusion, these results suggest ACSL4 is essential for tubular ferroptotic death during kidney fibrosis development and ACSL4 inhibition is a viable therapeutic approach to preventing fibrotic kidney diseases.

Novel aspect of neprilysin in kidney fibrosis via ACSL4-mediated ferroptosis of tubular epithelial cells.

Although inhibition of neprilysin (NEP) might be a therapeutic strategy with the potential to improve the outcome of chronic kidney disease (CKD), the versatile function of NEP with its mechanism remains obscure in kidney fibrosis. In the study, we found that NEP was abnormally increased in tubular epithelial cells of CKD patients, as well as unilateral ureteral obstruction and adenine diet-induced mice. Treatment with a United States Food and Drug Administration-approved NEP inhibitor Sacubitrilat (LBQ657) could alleviate ferroptosis, tubular injury, and delay the progression of kidney fibrosis in experimental mice. Similarly, genetic knockdown of NEP also inhibited tubular injury and fibrosis in transforming growth factor (TGF)-β1 -induced tubular cells. Mechanically, NEP overexpression aggravated the ferroptotic and fibrotic phenotype, which was restored by acyl-CoA synthetase long-chain family member 4 (ACSL4) knockdown. The NEP silencing attenuated TGF-β1-induced tubular cell ferroptosis and was exacerbated by ACSL4 overexpression. Collectively, for the first time, a novel aspect of NEP was explored in kidney fibrosis through ACSL4-mediated tubular epithelial cell ferroptosis. Our data further confirmed that NEP inhibition exerted a promising therapeutic against fibrotic kidney diseases.

#6755 NOVEL ASPECT OF NATURAL FLAVONOL FISETIN VIA INHIBITING ACSL4-MEDIATED TUBULAR FERROPTOSIS AGAINST FIBROTIC KIDNEY DISEASE

Abstract Background and Aims Kidney fibrosis is the hallmark of chronic kidney disease (CKD) progression, whereas no effective anti-fibrotic therapies exist. We previously reported that natural flavonol fisetin alleviated septic acute kidney injury and protected against hyperuricemic nephropathy in mice. However, the versatile role and potential mechanism of fisetin against fibrotic kidney disease have not been well characterized. Recently, tubular ferroptosis contributes to the pathogenesis of CKD with a persistent inflammatory and profibrotic response. Method Here, we found that fisetin significantly improved tubular injury, inflammation, and tubulointerstitial fibrosis in adenine diet- and unilateral ureteral obstruction (UUO)-induced CKD mice. The renoprotective effects of fisetin were also confirmed to suppress inflammatory response and fibrogenesis in adenine- or TGF-β1-stimulated tubular epithelial cells. Results Mechanistically, fisetin inhibited ferroptosis in the kidneys of CKD mice as well as in injured tubular epithelial cells, as evidenced by a decrease of ACSL4, COX2, HMGB1, and an increase of GPX4. Meanwhile, fisetin effectively restored ultrastructural abnormalities of mitochondrial morphology and relieved the elevated iron, the downregulated GSH and GSH/GSSG, as well as the upregulated lipid peroxide MDA in the kidneys of CKD mice. Notably, abnormal expression of ferroptosis key marker ACSL4 was verified in renal tubules of CKD patients (IgAN, MN, FSGS, LN, and DN) and mice (adenine and UUO), which was also observed in injured tubular cells. Furthermore, ACSL4 knockdown inhibited tubular ferroptosis, while ACSL4 overexpression blocked anti-ferroptotic effect of fisetin and reversed the cytoprotective, anti-inflammation, and anti-fibrosis of fisetin in injured tubular cells. Conclusion In summary, we for the first time explored a novel aspect of fisetin via inhibiting ACSL4-mediated tubular ferroptosis against fibrotic kidney diseases.

#5591 CIRCULATING ACTIVIN A REFLECTS THE SEVERITY OF RENAL FIBROSIS IN BIOPSY-PROVEN KIDNEY DISEASES—THE TAIPEI RENAL BIOPSY COHORT STUDY

Abstract Background and Aims Despite considerable evidence indicating circulating activin A as a novel renal biomarker, its performance for predicting the severity of renal fibrosis and major adverse renal events (MARE) has not yet been extensively studied. Method We sought to assess the relationship between plasma activin A, renal fibrosis severity, and incidence of MARE in 339 patients undergoing clinically indicated native renal biopsies. To determine the primary source of circulating activin A, RNA-sequencing and histological analyses were performed on kidney biopsy specimens from patients with chronic kidney disease (CKD). We also conducted in vitro experiments to investigate whether inhibiting endogenous activin A could attenuate TGF-β-mediated activation of cultured fibroblasts. Results The median baseline eGFR and proteinuria were 36 mL/min/1.73 m2 and 2.9 mg/mg creatinine, respectively. After multivariable adjustment, elevated plasma activin A was associated with the extent of renal fibrosis. Histological analysis showed increased activin A expression in kidney tissues from patients with CKD, mainly in interstitial myofibroblasts. RNA-sequencing of tubulointerstitial tissue from human biopsy samples also revealed a direct correlation between tissue activin A mRNA expression and plasma activin A levels. During a median follow-up of 22 months, 113 participants suffered MARE. Cox proportional hazards analysis revealed that plasma activin A was associated with higher risk of renal events; however, the association became insignificant after correcting for confounders. Results from in vitro studies demonstrated that knocking down activin A expression could prevent TGF-β-induced activation of NRK49F fibroblasts. Conclusion The findings of this study support activin A as a potential diagnostic and therapeutic target in fibrotic kidney disease.

#6741 TRANSCRIPTION FACTOR ATF3 EXACERBATES RENAL FIBROSIS THROUGH RECRUITING HISTONE ACETYLASES

Abstract Background and Aims Chronic kidney disease (CKD) threatens public health around the world. Renal fibrosis is a final common pathology in renal diseases of different etiologies. However, no efficient, broadly applicable anti-fibrotic therapies exist. Histone acetylation is an epigenetic modification involved in the regulation of multiple disease-specific gene expressions, balanced by histone acetyltransferases and histone deacetylase (HDAC) enzymes. Transcription factors (TF) could recruit histone-modifying enzymes and alter gene expression. But now, much remains unknown about the role of histone acetylation and whether there exits the regulator of it in fibrosis of CKD. Here, we for the first time explored a novel aspect of ATF3 promoting kidney fibrosis via recruiting the histone acetylases to fibrosis-related genes in CKD. Method We constructed a CKD mouse model by using adenine and oxonate. Serum were taken for the measurement of BUN and creatinine. Kidneys were harvested and processed for histological, sequencing, qRT-PCR, and western blotting analysis. RNA-seq and ChIP-seq (antibody: H3K27ac) of mouse kidneys were conducted for profiling the differential gene expression and H3K27ac. GO and KEGG analysis was performed for annotation of the function. Motif analysis was applied for searching the regulator of H3K27ac. Biopsy samples of patients with common CKD were used for immunohistochemical analysis. ATF3-KO mice were also conducted for defining the function of ATF3 in CKD. To profile the ATF3 binding site on chromatin, ChIP-seq of ATF3 in mice kidneys of normal control and CKD were also performed. Besides, the data contained ATF3 interact protein in databases (ChIP-atlas and BioGrid) and the results of protein interaction prediction were integrated for finding the protein-protein interaction of ATF3. TGF-β- stimulated TCMK-1 cell served as the cell model for further mechanism validation. CO-IP in cells clarifies that ATF3 could recruit the histone acetyltransferases. ChIP-qPCR of the fibrosis gene was performed to further verify the mechanism of ATF3-regulated gene expression. Results Firstly, compared with the normal control, histone modification H3K27ac repertoire was changed in chronic kidney disease. 11859 H3K27ac peaks specific to CKD, and 14996 H3K27ac peaks specific to healthy mice kidneys were found. The binding intensity of these regions has altered dramatically in the kidney of CKD. Pearson correlation analysis and function annotation showed that CKD-specific changes of H3K27ac were associated with fibrosis gene expression. Motif analysis showed that ATF3 in the top 10 TFs that may interact with H3K27ac. Among those TFs, ATF3 elicited the highest expression. Besides, ATF3 could co-localize with H3K27ac along the mice genome globally. By immunohistochemical staining analysis, we observed the upregulation of ATF3 in the kidneys of patients with biopsy-proven CKD. We found that knocking out the ATF3 could significantly reduce kidney damage in CKD mice. In parallel with improved morphological injuries, the level of kidney fibrosis gene expression was also significantly reduced in ATF3–/– mice. Furthermore, the H3K27ac on the mice chromatin altered along with the knockout of ATF3, and several fibrosis-related gene expressions which elevated in CKD were decreased by deficiency of ATF3. On the region of gene cd2, H3K27ac disappeared with the knockout of ATF3 in CKD. We found that ATF3 could interact with histone acetyltransferases (CBP, KAT7, P300) to promote the H3K27ac. Further evidenced by ChIP-qPCR of fibrosis gene cd2, we found that ATF3 could recruit the histone acetyltransferases on the fibrosis gene to promote the H3K27ac to provoke the gene expression in the cell model. Conclusion We profiled the landscape of H3K27ac in CKD, explored the epigenetic regulator of H3K27ac, and revealed that ATF3 promotes renal fibrosis in CKD. Notably, we for the first time explored a novel mechanism of ATF3 about recruiting histone acetyltransferases to regulate the H3K27ac on fibrosis gene. Our data highlighted that ATF3 might represent a potential therapeutic target against fibrotic kidney diseases.

Calponin 2 harnesses metabolic reprogramming to determine kidney fibrosis

ObjectiveIn the fibrotic kidneys, the extent of a formed deleterious microenvironment is determined by cellular mechanical forces. This process requires metabolism for energy. However, how cellular mechanics and metabolism are connected remains unclear. MethodsA multi-disciplinary approach was employed: the fibrotic kidney disease models were induced by renal ischemia-reperfusion injury and unilateral ureteral obstruction in Calponin 2 (CNN2) knockdown mice. Proteomics, bioinformatics, and in vivo and in vitro molecular experimental pathology studies were performed. ResultOur proteomics revealed that actin filament binding and cell metabolism are the two most dysregulated events in the fibrotic kidneys. As a prominent actin stabilizer, CNN2 was predominantly expressed in fibroblasts and pericytes. In CKD patients, CNN2 levels was markedly induced in blood. In mice, CNN2 knockdown preserves kidney function and alleviates fibrosis. Global proteomics profiled that CNN2 knockdown enhanced the activities of the key rate-limiting enzymes and regulators of fatty acid oxidation (FAO) in the diseased kidneys. Inhibiting carnitine palmitoyltransferase 1α in the FAO pathway resulted in lipid accumulation and extracellular matrix deposition in the fibrotic kidneys, which were restored after CNN2 knockdown. Bioinformatics and chromatin immunoprecipitation showed that CNN2 interactor, estrogen receptor 2 (ESR2), binds peroxisome proliferator-activated receptor-α (PPARα) to transcriptionally regulate FAO downstream target genes expression amid kidney fibrosis. In vitro, ESR2 knockdown repressed the mRNA levels of PPARα and the key genes in the FAO pathway. Conversely, activation of PPARα reduced CNN2-induced matrix inductions. ConclusionsOur results suggest that balancing cell mechanics and metabolism is crucial to develop therapeutic strategies to halt kidney fibrosis.

Mitochondrial micropeptide MOXI promotes fibrotic gene transcription by translocation to the nucleus and bridging N-acetyltransferase 14 with transcription factor c-Jun

Progressive fibrosis is a hallmark of chronic kidney disease, but we lack effective treatments to halt this destructive process. Micropeptides (peptides of no more than 100 amino acids) encoded by small open reading frames represent a new class of eukaryotic regulators. Here, we describe that the micropeptide regulator of β-oxidation (MOXI) regulates kidney fibrosis. MOXI expression was found to be up-regulated in human fibrotic kidney disease, and this correlated with the degree of fibrosis and loss of kidney function. MOXI was expressed in the cytoplasm and mitochondria of cultured tubular epithelial cells and translocated to the nucleus upon Transforming Growth Factor-β1 stimulation. Deletion of Moxi protected mice against fibrosis and inflammation in the folic acid and unilateral ureteral obstruction models. As a potential molecular therapy, treatment with an antisense MOXI oligonucleotide effectively knocked-down MOXI expression and protected against kidney fibrosis in both models. Bimolecular fluorescence complementation identified the enzyme N-acetyltransferase 14 (Nat14) and transcription factor c-Jun as MOXI binding partners. The MOXI/Nat14/c-Jun complex enhances basal and Transforming Growth Factor-β1 induced collagen I gene promoter activity. Phosphorylation at T49 is required for MOXI nuclear localization and for complex formation with Nat14 and c-Jun. Furthermore, mice with a MoxiT49A point mutation were protected in the models of kidney fibrosis. Thus, our studies demonstrate a key role for the micropeptide MOXI in kidney fibrosis and identify a new function of MOXI in forming a transcriptional complex with Nat14 and c-Jun.

Insulin-like growth factor 2 mRNA-binding protein 3 promotes kidney injury by regulating β-catenin signaling.

Wnt/β-catenin is a developmental signaling that plays a crucial role in driving kidney fibrosis after injury. Activation of β-catenin is presumed to be regulated through the post-translational protein modification. Little is known about whether β-catenin is also subjected to regulation at the post-transcriptional mRNA level. Here, we report that insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3) plays a pivotal role in regulating β-catenin. IGF2BP3 was upregulated in renal tubular epithelium of various animal models and patients with chronic kidney disease. IGF2BP3 not only was a direct downstream target of Wnt/β-catenin but also obligatory for transducing Wnt signal. In vitro, overexpression of IGF2BP3 in kidney tubular cells induced fibrotic responses, whereas knockdown of endogenous IGF2BP3 prevented the expression of injury and fibrosis markers in tubular cells after Wnt3a stimulation. In vivo, exogenous IGF2BP3 promoted β-catenin activation and aggravated kidney fibrosis, while knockdown of IGF2BP3 ameliorated renal fibrotic lesions after obstructive injury. RNA immunoprecipitation and mRNA stability assay revealed that IGF2BP3 directly bound to β-catenin mRNA and stabilized it from degradation. Furthermore, knockdown of IGF2BP3 in tubular cells accelerated β-catenin mRNA degradation in vitro. These studies demonstrate that IGF2BP3 promotes β-catenin signaling and drives kidney fibrosis, which may be mediated through stabilizing β-catenin mRNA. Our findings uncover a new dimension of the complex regulation of Wnt/β-catenin signaling and suggest a potential target for therapeutic intervention of fibrotic kidney diseases.

Targeted inhibition of TGF-β type I receptor by AZ12601011 protects against kidney fibrosis

Renal fibrosis, a common feature of chronic kidney disease, causes the progressive loss of renal function, in which TGF-β1 plays a critical role. In this study, we found that expression levels of TGF-β1 and its receptor 1 (TGF-βR1) were both significantly increased in obstructive fibrosis kidneys. AZ12601011 is a small molecular inhibitor of TGF-βR1; however, its therapeutic potential for renal fibrosis remains unclear. During the experiments, AZ12601011 was applied to various models of renal fibrosis followed by unilateral ureteral obstruction (UUO) and ischemia/reperfusion (I/R) in vivo, in addition to renal tubular epithelial cells (TECs) challenged by hypoxia/reoxygenation (H/R) and TGF-β1in vitro. Our results revealed that AZ12601011 ameliorated renal injuries and fibrosis shown by PAS, HE, and Masson staining, which was consistent with the decrease in Col-1 and α-SMA expression in the kidneys from UUO and I/R mice. Similarly, in vitro data showed that AZ12601011 inhibited the induction of Col-1 and α-SMA in both TECs treated with TGF-β1 and H/R. In addition, the results of cellular thermal shift assay (CETSA), molecular docking, and western bolt indicated that AZ12601011 could directly bind to TGF-βR1 and block activation of the downstream Smad3. Taken together, our findings suggest that AZ12601011 can attenuate renal fibrosis by blocking the TGF-β/Smad3 signaling pathway and it might serve as a promising clinical candidate in the fight against fibrotic kidney diseases.

Pharmacological and Genetic Inhibition of HDAC4 Alleviates Renal Injury and Fibrosis in Mice.

Histone deacetylase 4 (HDAC4) has been shown to be involved in cell proliferation, differentiation, and migration and is associated with a variety of cancers. However, the role of HDAC4 in renal fibrogenesis and its mechanisms are unclear. We assessed the role of HDAC4 and possible mechanisms of fibrosis in a murine model of kidney injury induced by unilateral ureteral obstruction (UUO) using tasquinimod, a highly selective HDAC4 inhibitor, and knockout mice with depletion of HDAC4 in renal tubular cells. UUO injury resulted in increased expression of HDAC4 and fibrotic proteins fibronectin and α-smooth muscle actin, while treatment with tasquinimod or knockout of HDAC4 significantly reduced their expression. Pharmacological and genetic inhibition of HDAC4 also decreased tubular epithelial cell arrest in the G2/M phase of the cell cycle, expression of transforming growth factor-β1 and phosphorylation of Smad3, signal transducer and activator of transcription 3, and extracellular signal-regulated kinase 1/2 in the injured kidney. Moreover, tasquinimod treatment or HDAC4 deletion inhibited UUO-induced renal tubular cell injury and apoptosis as indicated by reduced expression of neutrophil gelatinase–associated lipocalin, Bax, and inhibition of caspase-3. Finally, administration of tasquinimod or knockdown of HDAC4 prevented injury-related repression of Klotho, a renoprotective protein. Our results indicate that HDAC4 is critically involved in renal tubular injury and fibrosis and suggest that HDAC4 is a potential therapeutic target for treatment of chronic fibrotic kidney disease.

Mitochondrial interaction of fibrosis-protective 5-methoxy tryptophan enhances collagen uptake by macrophages

5-methoxy tryptophan (5-MTP) is an anti-fibrotic metabolite made by fibroblasts and epithelial cells, present in a micromolar concentrations in human blood, and is associated with the progression of fibrotic kidney disease, but the mechanism is unclear. Here, we show by microscopy and functional assays that 5-MTP influences mitochondria in human peripheral blood monocyte-derived macrophages. As a result, the mitochondrial membranes are more rigid, more branched, and are protected against oxidation. The macrophages also change their metabolism by reducing mitochondrial import of acyl-carnitines, intermediates of fatty acid metabolism, driving glucose import. Moreover, 5-MTP increases the endocytosis of collagen by macrophages, and experiments with inhibition of glucose uptake showed that this is a direct result of their altered metabolism. However, 5-MTP does not affect the macrophages following pathogenic stimulation, due to 5-MTP degradation by induced expression of indole-amine oxygenase-1 (IDO-1). Thus, 5-MTP is a fibrosis-protective metabolite that, in absence of pathogenic stimulation, promotes collagen uptake by anti-inflammatory macrophages by altering the physicochemical properties of their mitochondrial membranes.

Histone Acetylation and Modifiers in Renal Fibrosis.

Histones are the most abundant proteins bound to DNA in eukaryotic cells and frequently subjected to post-modifications such as acetylation, methylation, phosphorylation and ubiquitination. Many studies have shown that histone modifications, especially histone acetylation, play an important role in the development and progression of renal fibrosis. Histone acetylation is regulated by three families of proteins, including histone acetyltransferases (HATs), histone deacetylases (HDACs) and bromodomain and extraterminal (BET) proteins. These acetylation modifiers are involved in a variety of pathophysiological processes leading to the development of renal fibrosis, including partial epithelial-mesenchymal transition, renal fibroblast activation, inflammatory response, and the expression of pro-fibrosis factors. In this review, we summarize the role and regulatory mechanisms of HATs, HDACs and BET proteins in renal fibrosis and provide evidence for targeting these modifiers to treat various chronic fibrotic kidney diseases in animal models.

A Neutralizing IL-11 Antibody Improves Renal Function and Increases Lifespan in a Mouse Model of Alport Syndrome.

Alport syndrome is a genetic disorder characterized by a defective glomerular basement membrane, tubulointerstitial fibrosis, inflammation, and progressive renal failure. IL-11 was recently implicated in fibrotic kidney disease, but its role in Alport syndrome is unknown. We determined IL-11 expression by molecular analyses and in an Alport syndrome mouse model. We assessed the effects of a neutralizing IL-11 antibody (×203) versus an IgG control in Col4a3-/- mice (lacking the gene encoding a type IV collagen component) on renal tubule damage, function, fibrosis, and inflammation. Effects of ×203, the IgG control, an angiotensin-converting enzyme (ACE) inhibitor (ramipril), or ramipril+X203 on lifespan were also studied. In Col4a3-/- mice, as kidney failure advanced, renal IL-11 levels increased, and IL-11 expression localized to tubular epithelial cells. The IL-11 receptor (IL-11RA1) is expressed in tubular epithelial cells and podocytes and is upregulated in tubular epithelial cells of Col4a3-/- mice. Administration of ×203 reduced albuminuria, improved renal function, and preserved podocyte numbers and levels of key podocyte proteins that are reduced in Col4a3-/- mice; these effects were accompanied by reduced fibrosis and inflammation, attenuation of epithelial-to-mesenchymal transition, and increased expression of regenerative markers. X203 attenuated pathogenic ERK and STAT3 pathways, which were activated in Col4a3-/- mice. The median lifespan of Col4a3-/- mice was prolonged 22% by ramipril, 44% with ×203, and 99% with ramipril+X203. In an Alport syndrome mouse model, renal IL-11 is upregulated, and neutralization of IL-11 reduces epithelial-to-mesenchymal transition, fibrosis, and inflammation while improving renal function. Anti-IL-11 combined with ACE inhibition synergistically extends lifespan. This suggests that a therapeutic approach targeting IL-11 holds promise for progressive kidney disease in Alport syndrome.

Targeting CC chemokine ligand (CCL) 20 by miR-143-5p alleviate lead poisoning-induced renal fibrosis by regulating interstitial fibroblasts excessive proliferation and dysfunction

ABSTRACT Environmental lead contamination can cause chronic renal disease with a common clinical manifestation of renal fibrosis and constitutes a major global public health threat. Aberrant proliferation and extracellular matrix (ECM) accumulation in renal interstitial fibroblasts are key pathological causes of renal fibrosis. However, the mechanism underlying lead-induced kidney fibrosis remains unclear. The present study analyzed gene expression prolifes in lead acetate-treated primary mice renal interstitial fibroblasts and confirmed the aberrant expression of CC chemokine ligand (CCL) 20, one of the most obvious up-regulated genes. Analogously, lead acetate exposure dose-dependently increased CCL20 transcription, protein expression and release. Knockdown of CCL20 suppressed lead acetate-induced fibroblast proliferation, hydroxyproline contents, transforming growth factor-beta production and ECM-related protein (Collagen I and fibronectin) expression. Bioinformatics analysis predicted five top miRNAs targeting CCL20. Among them, miR-143-5p expression was dose-dependently decreased in lead acetate-treated fibroblasts. Mechanistically, miR-143-5p directly targeted CCL20. Elevation of miR-143-5p antagonized lead acetate-induced fibroblast proliferation, hydroxyproline and ECM-related protein expression, which were reversed by CCL20 overexpression. Additionally, CCL20 knockdown suppressed lead acetate-mediated Smad2/3 and AKT pathway activation. Notably, miR-143-5p overexpression attenuated the activation of the Smad2/3 and AKT pathway in lead acetate-exposed fibroblasts, which was counteracted by CCL20 elevation. miR-143-5p injection ameliorated renal fibrosis progression in mice in vivo. Thus, targeting CCL20 by miR-143-5p could alleviate renal fibrosis progression by regulating fibroblast proliferation and ECM deposition via the Smad2/3 and AKT signaling, providing a potential therapeutic target for environmental lead contamination-evoked fibrotic kidney disease.