Interstitial Fibroblast Proliferation Research Articles

Oxidatively stressed extracellular microenvironment drives fibroblast activation and kidney fibrosis

Kidney fibrosis is associated with tubular injury, oxidative stress and activation of interstitial fibroblasts. However, whether these events are somehow connected is poorly understood. In this study, we show that glutathione peroxidase-3 (GPX3) depletion in renal tubular epithelium after kidney injury plays a central role in orchestrating an oxidatively stressed extracellular microenvironment, which drives interstitial fibroblast activation and proliferation. Through transcriptional profiling by RNA-sequencing, we found that the expression of GPX3 was down-regulated in various models of chronic kidney disease (CKD), which was correlated with induction of nicotinamide adenine dinucleotide phosphate (NAPDH) oxidase-4 (NOX4). By using decellularized extracellular matrix (ECM) scaffold, we demonstrated that GPX3-depleted extracellular microenvironment spontaneously induced NOX4 expression and reactive oxygen species (ROS) production in renal fibroblasts and triggered their activation and proliferation. Activation of NOX4 by advanced oxidation protein products (AOPPs) mimicked the loss of GPX3, increased the production of ROS, stimulated fibroblast activation and proliferation, and activated protein kinase C-α (PKCα)/mitogen-activated protein kinase (MAPK)/signal transducer and activator of transcription 3 (STAT3) signaling. Silencing NOX4 or inhibition of MAPK with small molecule inhibitors hampered fibroblast activation and proliferation. In mouse model of CKD, knockdown of NOX4 repressed renal fibroblast activation and proliferation and alleviated kidney fibrosis. These results indicate that loss of GPX3 orchestrates an oxidatively stressed extracellular microenvironment, which promotes fibroblast activation and proliferation through a cascade of signal transduction. Our studies underscore the crucial role of extracellular microenvironment in driving fibroblast activation and kidney fibrosis.

The Perspective of Vitamin D on suPAR-Related AKI in COVID-19.

The coronavirus disease 2019 (COVID-19) pandemic has claimed the lives of millions of people around the world. Severe vitamin D deficiency can increase the risk of death in people with COVID-19. There is growing evidence that acute kidney injury (AKI) is common in COVID-19 patients and is associated with poorer clinical outcomes. The kidney effects of SARS-CoV-2 are directly mediated by angiotensin 2-converting enzyme (ACE2) receptors. AKI is also caused by indirect causes such as the hypercoagulable state and microvascular thrombosis. The increased release of soluble urokinase-type plasminogen activator receptor (suPAR) from immature myeloid cells reduces plasminogen activation by the competitive inhibition of urokinase-type plasminogen activator, which results in low plasmin levels and a fibrinolytic state in COVID-19. Frequent hypercoagulability in critically ill patients with COVID-19 may exacerbate the severity of thrombosis. Versican expression in proximal tubular cells leads to the proliferation of interstitial fibroblasts through the C3a and suPAR pathways. Vitamin D attenuates the local expression of podocyte uPAR and decreases elevated circulating suPAR levels caused by systemic inflammation. This decrease preserves the function and structure of the glomerular barrier, thereby maintaining renal function. The attenuated hyperinflammatory state reduces complement activation, resulting in lower serum C3a levels. Vitamin D can also protect against COVID-19 by modulating innate and adaptive immunity, increasing ACE2 expression, and inhibiting the renin–angiotensin–aldosterone system. We hypothesized that by reducing suPAR levels, appropriate vitamin D supplementation could prevent the progression and reduce the severity of AKI in COVID-19 patients, although the data available require further elucidation.

Tubule-derived INHBB promotes interstitial fibroblast activation and renal fibrosis.

Upstream stimuli for myofibroblast activation are of considerable interest for understanding the mechanisms underlying renal fibrosis. Activin B, a member of the TGF-β family, exists as a homodimer of inhibin subunit beta B (INHBB), but its role in renal fibrosis remains unknown. We found that INHBB expression was significantly increased in various renal fibrosis models and human chronic kidney disease specimens with renal fibrosis. Notably, the increase of INHBB occurred mainly in the tubular epithelial cells (TECs). In vivo, inhibiting INHBB blocked the activation of interstitial fibroblasts and ameliorated the renal fibrosis induced by unilateral ureteral obstruction or ischemia-reperfusion injury, while ectopic expression of INHBB in the TECs was able to activate interstitial fibroblasts and initiate interstitial fibrosis. In vitro, overexpression of INHBB in TECs led to the secretion of activin B, thereby promoting the proliferation and activation of interstitial fibroblasts through activin B/Smad signaling. Furthermore, inhibition of activin B/Smad signaling attenuated the fibrotic response caused by tubular INHBB. Mechanistically, the upregulation of INHBB depended on the transcription factor Sox9 in the injured TECs. Clinical analyses also identified a positive correlation between Sox9 and INHBB expression in human specimens, suggesting the Sox9/INHBB axis as a positive regulator of renal fibrosis. In conclusion, tubule-derived INHBB is implicated in the pathogenesis of renal fibrosis by activating the surrounding fibroblasts in a paracrine manner, thereby exhibiting as a potential therapeutic target. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Critical roles of SMYD2 lysine methyltransferase in mediating renal fibroblast activation and kidney fibrosis.

SET and MYND domain protein 2 (SMYD2) is a lysine methyltransferase that mediates histone H3 lysine 36 trimethylation (H3K36me3) and acts as a regulator of tumorgenesis and cystic growth. However, its role in renal fibrosis remains unknown. In this study, we found that SMYD2 was highly expressed in the murine kidney of renal fibrosis induced by unilateral ureteral obstruction, and primarily located in interstitial fibroblasts and renal tubular epithelial cells. Pharmacological inhibition of SMYD2 with AZ505, a highly selective inhibitor of SMYD2, protected against renal fibrosis and inhibited activation/proliferation of renal interstitial fibroblasts and conversion of epithelial cells to a profibrotic phenotype in this model. In cultured renal interstitial fibroblasts, treatment with AZ505 or silencing of SMYD2 by specific siRNA also inhibited serum- or TGF-β1-induced activation and proliferation of renal interstitial fibroblasts. Mechanistic studies showed that SMYD2 inhibition reduced phosphorylation of several profibrotic signaling molecules, including Smad3, extracellular signal-regulated kinase 1/2, AKT, signal transducer and activator of transcription-3 and nuclear factor-κB in both injured kidney and cultured renal fibroblasts. AZ505 was also effective in suppressing renal expression of Snail and Twist, two transcriptional factors that mediate renal partial epithelial-mesenchymal transition and fibrosis. Conversely, AZ505 treatment prevented downregulation of Smad7, a renoprotective factor in vivo and in vitro. These results indicate that SMYD2 plays a critical role in mediating conversion of epithelial cells to a profibrotic phenotype, renal fibroblast activation and renal fibrogenesis, and suggest that SMYD2 may be a potential target for the treatment of chronic fibrosis in kidney disease.

Mesenchymal stem cell-derived extracellular vesicles suppress the fibroblast proliferation by downregulating FZD6 expression in fibroblasts via micrRNA-29b-3p in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF), a progressive and fatal lung disease, usually leads to an irreversible distortion of the pulmonary structure. The functional roles of bone marrow-derived mesenchymal stem cells (BMSC)-secreted extracellular vesicles (EVs) in fibroblasts have been implicated, yet their actions in the treatment of IPF are not fully understood. This study investigated the roles of BMSC-derived EVs expressing miR-29b-3p in fibroblasts in IPF treatment. EVs derived from BMSCs were successfully isolated and could be internalized by pulmonary fibroblasts, and Cell Counting Kit-8 (CCK-8) and Transwell assay results identified that EVs inhibited the activation of fibroblast in IPF. miR-29b-3p, frizzled 6 (FZD6), α-skeletal muscle actin (α-SMA), and Collagen I expressions were examined, which revealed that miR-29b-3p was poorly expressed and FZD6, α-SMA, and Collagen I were overexpressed in pulmonary tissues. Dual-luciferase reporter assay results demonstrated that miR-29b-3p could inversely target FZD6 expression. The gain- and loss-of-function assays were conducted to determine regulatory effects of FZD6 and miR-29b-3p on IPF. CCK-8 and Transwell assays results displayed that BMSCs-derived EVs overexpressing miR-29b-3p contributed to inhibited pulmonary interstitial fibroblast proliferation, migration, invasion, and differentiation. Furthermore, the effects of BMSCs-derived EVs overexpressing miR-29b-3p on IPF progression were assessed in vivo, which confirmed the repressive effects of BMSCs-derived EVs overexpressing miR-29b-3p on IPF progression. Collectively, BMSCs-derived EVs overexpressing miR-29b-3p relieve IPF through FZD6.

Melatonin Prevents Transforming Growth Factor-β1-Stimulated Transdifferentiation of Renal Interstitial Fibroblasts to Myofibroblasts by Suppressing Reactive Oxygen Species-Dependent Mechanisms.

Accumulating evidence suggests that the pineal hormone melatonin displays protective effects against renal fibrosis, but the mechanisms remain poorly understood. Here, we investigate the effect of the pineal hormone on transdifferentiation of renal fibroblasts to myofibroblasts invoked by transforming growth factor-β1 (TGF-β1). Increased proliferation and activation of renal interstitial fibroblasts after TGF-β1 treatment were attenuated by melatonin pretreatment. Mechanistically, melatonin suppressed Smad2/3 phosphorylation and nuclear co-localization of their phosphorylated forms and Smad4 after TGF-β1 stimulation. In addition, increased phosphorylations of Akt, extracellular signal-regulated kinase 1/2, and p38 after TGF-β1 treatment were also suppressed by the hormone. These effects of melatonin were not affected by pharmacological and genetic inhibition of its membrane receptors. Furthermore, melatonin significantly reversed an increase of intracellular reactive oxygen species (ROS) and malondialdehyde levels, and a decrease of the reduced glutathione/oxidized glutathione ratio after TGF-β1 treatment. Finally, TGF-β1-induced proliferation and activation were also suppressed by N-acetylcysteine. Altogether, these findings suggest that the pineal hormone melatonin prevents TGF-β1-induced transdifferentiation of renal interstitial fibroblasts to myofibroblasts via inhibition of Smad and non-Smad signaling cadcades by inhibiting ROS-mediated mechanisms in its receptor-independent manner.

Silenced miR-21 inhibits renal interstitial fibrosis via targeting ERK1/2 signaling pathway in mice.

To study the influence of micro ribonucleic acid (miR)-21 on the renal interstitial fibrosis (RIF) model mice, and to preliminarily elucidate the mechanism of action of miR-21 in the development of RIF by studying the influences of miR-21 on the expressions of the proteins related to the extracellular signal-regulated kinase (ERK) 1/2 signaling pathway and its downstream proteins. The mouse model of the left unilateral ureteral obstruction (UUO) was established. The experimental mice were divided into the Sham group and UUO group and were normally fed for 3 weeks. Then, they were executed, and their blood was extracted to determine the renal function-related indicators. The left kidney was excised, and the corresponding specimens were reserved for observing the appearance of the kidney and the morphology of the renal tubules and interstitium. The relative expression levels of epithelial (E-)cadherin, α-smooth muscle actin (α-SMA), and ERK1/2 phosphorylated ERK1/2 (p-ERK1/2), the transforming growth factor-β1 (TGF-β1) and the connective tissue growth factor (CTGF) proteins in renal tissues were determined. After the human renal proximal tubular epithelial cell line, the human kidney-2 (HK-2) was treated with high glucose (HG) combined with silenced miR-21 or the ERK1/2 inhibitor PD98059, the relative expression levels of ɑ-SMA and TGF-β1 protein were measured. UUO group had significantly higher content of blood urea nitrogen (BUN), serum creatinine (SCr), and uric acid (UA) than the Sham group, and exhibited the infiltration of renal interstitial monocytes and lymphocytes, renal tubular podocyte damage, phenotypic transformation and atrophy, the activation and proliferation of interstitial fibroblasts, and excessive deposition of extracellular matrix (ECM). Moreover, the expression level of E-cadherin in the renal tissues was decreased, but the relative expression levels of ɑ-SMA, and TGF-β1, CTGF, and p-ERK1/2 proteins were evidently elevated. Lower relative expression levels of ɑ-SMA and TGF-β1 protein were detected in the human renal proximal tubular epithelial cell line HK-2 after the combined treatment with HG and silenced miR-21 or the ERK1/2 inhibitor PD98059. MiR-21 may be related to the occurrence and development of RIF. Silenced miR-21 probably suppresses RIF via the ERK1/2 signaling pathway.

MicroRNA-328 in exosomes derived from M2 macrophages exerts a promotive effect on the progression of pulmonary fibrosis via FAM13A in a rat model

Currently, exosome-enclosed microRNAs (miRs) in exhaled breath have potential for biomarker discovery in patients with pulmonary diseases. This study was performed to investigate the roles of M2 macrophage-derived exosomes expressing miR-328 in pulmonary fibrosis (PF). Microarray-based analysis was used to screen differentially expressed genes (DEGs) and regulatory miRs in PF. The miR-target relationship between FAM13A and miR-328 was confirmed. The expression of FAM13A and miR-328 was measured in PF rats, and gain- and loss-of-function assays were conducted to determine the regulatory effects of FAM13A and miR-328 on PF. In addition, exosomes derived from M2 macrophages were isolated and then cocultured with pulmonary interstitial fibroblasts to identify the role of these exosomes in PF. Furthermore, the effects of M2 macrophage-derived exosomes overexpressing miR-328 on pulmonary fibroblast proliferation and the progression of PF were assessed in vivo. miR-328 might perform a vital function in PF by regulating FAM13A. FAM13A expression was downregulated while miR-328 expression was upregulated in rats with PF, and a miR-target relationship between miR-328 and FAM13A was observed. Additionally, miR-328 overexpression and FAM13A silencing each were suggested to promote pulmonary interstitial fibroblast proliferation and the expression of Collagen 1A, Collagen 3A and α-SMA. Then, in vitro experiments demonstrated that M2 macrophage-derived exosomes overexpressing miR-328 contributed to enhanced pulmonary interstitial fibroblast proliferation and promoted PF. Furthermore, in vivo experiments confirmed the promotive effects of M2 macrophage-derived exosomes overexpressing miR-328 on the progression of PF. Collectively, the results showed that M2 macrophage-derived exosomes overexpressing miR-328 aggravate PF through the regulation of FAM13A.

The C5a/C5aR1 axis promotes progression of renal tubulointerstitial fibrosis in a mouse model of renal ischemia/reperfusion injury

C5a is a potent proinflammatory agonist that mediates renal ischemia reperfusion (IR) injury, but the potential for modulating chronic post-ischemic fibrosis and use of therapeutic antagonist are undefined. Here we determine whether C5a receptor 1 (C5aR1) signaling is essential to the development of post-ischemic fibrosis and if it is a valid target for therapeutic blockade with soluble receptor antagonist. C5aR1 is required for the development of renal tubulointerstitial fibrosis in a murine model of renal ischemia/reperfusion injury. Deficiency of C5aR1 protected mice from the development of the fibrosis. This protection was associated with attenuated deposition of extracellular matrix components (fibronectin, collagen I), reduced cellular infiltrates (CD45, F4/80), and gene expression of proinflammatory and profibrogenic mediators in the kidney. In an invitro model of hypoxia/reoxygenation, C5a stimulation caused renal fibroblast proliferation and activation, and upregulated gene expression of interleukin-1α (IL-1α), IL-6 and transforming growth factor-α (TGF-α) in renal tubular epithelial cells and monocytes/macrophages. Administration of a C5aR1 antagonist (PMX53) significantly reduced renal injury and tubulointerstitial fibrosis. Thus, our results demonstrate a pathogenic role for C5aR1 in the progression of tubulointerstitial fibrosis following renal IR injury and support that C5aR1-mediated local inflammatory responses to hypoxic renal injury contribute to tubulointerstitial fibrosis through several cellular pathways, namely, promoting tubule injury, interstitial fibroblast proliferation and epithelial-to-mesenchymal transition of renal tubular epithelial cells. Our results also suggest the C5a-C5aR1 interaction is a therapeutic target for chronic post-ischemic fibrosis.

Characterization of septin expression in normal and fibrotic kidneys.

Chronic kidney disease (CKD) is characterized by the loss of nephrons and worsening organ-fibrosis that leads to deterioration and ultimately the total breakdown of kidney function. Renal fibrosis has become a major public health problem worldwide and necessitates hemodialysis and kidney transplantation in affected patients. CKD is mainly characterized by the activation and proliferation of interstitial fibroblasts and by excessive synthesis and accumulation of extracellular matrix components, causing the disruption of the normal tissue architecture of the kidney. Septins are GTPase proteins associated with membranes, actin filaments, and microtubules and are undoubtedly crucial for cytoskeleton organization. Although some septins are involved in liver fibrosis, they have not been investigated in the context of renal fibrosis. Here, we show that numerous septins are expressed in the healthy kidney and demonstrate in fibrotic mouse kidneys that various septins are remarkably up-regulated in the tubulointerstitium compared to contralateral control kidneys. We observed the same findings in human fibrotic kidneys. In both healthy and fibrotic kidneys, septins are coexpressed with extracellular matrix components, reinforcing the structural function of septins as cytoskeletal components. Furthermore, we could show in septin 8-deficient mice that septin 8 is dispensable for the formation of renal fibrosis, and that no other septin was compensatory changed in kidneys compared to wild-type mice.

β-Aminoisobutyric acid ameliorates the renal fibrosis in mouse obstructed kidneys via inhibition of renal fibroblast activation and fibrosis

Renal fibrosis is a hallmark feature of chronic kidney disease, which is reflected by proliferation and migration of interstitial fibroblasts and extracellular matrix (ECM) accumulation. β-Aminoisobutyric acid (BAIBA) is recently demonstrated to exert a protective role from metabolic diseases. However, whether and how BAIBA on fibroblast activation and renal fibrosis response to angiotensin II (Ang II) remains largely obscure. Herein, we showed that BAIBA significantly depressed the proliferation and migration of NRK-49F cells in vitro. Treatment with Ang II remarkably up-regulated the expressions of fibronectin (FN), collagen 1 (COL 1), α-smooth muscle actin (α-SMA), interleukin-17 (IL-17) and nicotinamide adenine dinucleotide phosphate oxidase (NOX2)-derived reactive oxygen species (ROS) production in cultured NRK-49F cells. Pretreatment with BAIBA almost blocked Ang II-induced ECM production and IL-17-mediated oxidative stress in NRK-49F cells. BAIBA treatment ameliorates fibroblasts activation and renal fibrosis in rat obstructed kidneys involving inhibition of Ang II/IL-17/ROS signaling transduction, which may be considered as a therapeutic candidate for fibrosis-related diseases.

Tenascin-C Is a Major Component of the Fibrogenic Niche in Kidney Fibrosis.

Kidney fibrosis initiates at certain focal sites in which the fibrogenic niche provides a specialized microenvironment that facilitates fibroblast activation and proliferation. However, the molecular identity of these fibrogenic niches is poorly characterized. Here, we determined whether tenascin-C (TNC), an extracellular matrix glycoprotein, is a component of the fibrogenic niche in kidney fibrosis. In vivo, TNC expression increased rapidly in kidneys subjected to unilateral ureteral obstruction or ischemia/reperfusion injury and predominantly localized at the foci rich in fibroblasts in renal interstitium. In vitro, TNC selectively promoted renal interstitial fibroblast proliferation, bromodeoxyuridine incorporation, and the expression of proliferation-related genes. The mitogenic activity of TNC required the integrin/focal adhesion kinase/mitogen-activated protein kinase signaling cascade. Using decellularized extracellular matrix scaffolds, we found that TNC-enriched scaffolds facilitated fibroblast proliferation, whereas TNC-deprived scaffolds inhibited proliferation. Matrix scaffold prepared from fibrotic kidney also promoted greater ex vivo fibroblast proliferation than did scaffolds prepared from healthy kidney. Conversely, small interfering RNA-mediated knockdown of TNC in vivo repressed injury-induced fibroblast expansion and renal fibrosis. These studies identify TNC as a major constituent of the fibrogenic niche that promotes fibroblast proliferation, and illustrate a pivotal role for the TNC-enriched microenvironment in kidney fibrogenesis.

IgG4-related meningeal disease

Objective To explore the clinical and pathological characteristics, diagnosis and differential diagnosis of IgG4-related meningeal disease. Methods and Results A 49-year-old male patient suffered from headache for nearly 2 years and the symptom was aggravated progressively for over one month. MRI revealed space-occupying lesion in left parietal lobe, with irregular signal and clear borderline. Contrast-enhanced MRI showed homogeneous enhancement, and obvious meningeal thicking just like dural tail sign. The patient underwent operation, and the lesion was totally removed. Histologically, it showed a large amount of hyperplastic collagen fibrous tissue with inflammatory cells, including a large number of plasmocytes, as well as scattered lymphocytes and a small number of eosinophilic granulocytes. Russell bodies which were homogenously positive for eosin could be seen in some plasmocytes. Focal necrosis, and proliferation of interstitial fibroblasts and small vessels were found. The lesion had no capsules and invaded surrounding tissues. Immunohistochemical staining showed the plasmocytes were diffusely positive for IgG and IgG4 (> 60%), and were positive for CD38 and CD138 on the membrane. Lymphocytes were positive for CD3, CD4 or CD20 on the membrane. The serum IgG4 level was 1.05 g/L. Final pathological diagnosis was IgG4-related meningeal disease in left parietal lobe. After operation, the patient received anti-infectious, anti-epileptic and nutrition support treatment, and the symptoms were markedly improved. The patient was discharged after 26 d, but was lost to follow-up. Conclusions IgG4-related meningeal disease is a rare disease. Due to the atypical imaging features, it may be difficult to differentiate IgG4-related meningeal disease from other diseases with prominent inflammatory cells and stromal fibrosis. The elevated serum IgG4 level may provide diagnostic cues. However, a definite diagnosis depends on characteristic histological and immunohistochemical features. DOI: 10.3969/j.issn.1672-6731.2016.03.010

Mitochondrial Pathology and Glycolytic Shift during Proximal Tubule Atrophy after Ischemic AKI.

During recovery by regeneration after AKI, proximal tubule cells can fail to redifferentiate, undergo premature growth arrest, and become atrophic. The atrophic tubules display pathologically persistent signaling increases that trigger production of profibrotic peptides, proliferation of interstitial fibroblasts, and fibrosis. We studied proximal tubules after ischemia-reperfusion injury (IRI) to characterize possible mitochondrial pathologies and alterations of critical enzymes that govern energy metabolism. In rat kidneys, tubules undergoing atrophy late after IRI but not normally recovering tubules showed greatly reduced mitochondrial number, with rounded profiles, and large autophagolysosomes. Studies after IRI of kidneys in mice, done in parallel, showed large scale loss of the oxidant-sensitive mitochondrial protein Mpv17L. Renal expression of hypoxia markers also increased after IRI. During early and late reperfusion after IRI, kidneys exhibited increased lactate and pyruvate content and hexokinase activity, which are indicators of glycolysis. Furthermore, normally regenerating tubules as well as tubules undergoing atrophy exhibited increased glycolytic enzyme expression and inhibitory phosphorylation of pyruvate dehydrogenase. TGF-β antagonism prevented these effects. Our data show that the metabolic switch occurred early during regeneration after injury and was reversed during normal tubule recovery but persisted and became progressively more severe in tubule cells that failed to redifferentiate. In conclusion, irreversibility of the metabolic switch, taking place in the context of hypoxia, high TGF-β signaling and depletion of mitochondria characterizes the development of atrophy in proximal tubule cells and may contribute to the renal pathology after AKI.

MicroRNA-21: a central regulator of fibrotic diseases via various targets.

MicroRNAs (miRNAs) are small non-coding RNA molecules that diversely regulate physiological and pathophysiological processes by specifically binding to different regions of targeting messenger RNAs (mRNAs). Fibrosis is characterized by the abnormal proliferation of fibroblasts and the deposition of the extracellular matrix (ECM). Both clinical and experimental animal studies have revealed that aberrant expression of miRNAs is closely associated with the development of fibrotic diseases. microRNA-21 (miR-21) is a ubiquitously expressed miRNA that is traditionally considered to be an oncogenic miRNA (oncomiR). Recent studies have demonstrated that elevated expression of miR-21 may play a vital role in the development of fibrosis by promoting the proliferation of interstitial fibroblasts and increasing the abnormal deposition of the ECM. In this review, we comprehensively summarize the role of miR-21 in tissue fibrosis. Furthermore, we highlight miR-21 as a potential diagnostic and prognostic marker and therapeutic target for fibrosis diseases.

Abstract 76: Meox1 is a Cell-cycle Oscillator Required for Mitotic Transition and Proliferation in Cardiac Fibroblasts

Background: Tissue fibrosis plays important roles in the pathogenesis of chronic diseases, including heart failure. The mechanism underlying interstitial fibroblast proliferation is a promising analytical target for therapeutic applications. Here we developed quantitative epigenome profiling to identify a critical regulator in interstitial cell populations that emerges during the progression of heart failure. Methods and Results: We subjected pressure-overloaded hearts of mice to trimethylated histone H3 lysine 4 (H3K4me3) ChIP-sequence and RNA-sequence. Expression analysis followed by quantitative H3K4me3 profiling identified 45 fibrosis-related genes with significant H3K4me3 enrichment in failing hearts, including Meox1 transcription factor. Meox1 emerged in the interstitial fibrotic region in failing heart, and intriguingly Meox1 was expressed in the limited population of cardiac fibroblasts both in vivo and in vitro. Meox1-positive fibroblasts were increased in response to a paracrine signal from cardiomyocytes, and knockdown of Meox1 completely inhibited the reactive proliferation of cardiac fibroblasts stimulated by conditioned medium from cardiomyocytes. Gene expression profiling combined with siRNAs clarified that Meox1 depletion resulted in down regulation in the mitosis-related genes including Aurora B kinase. Indeed, Meox1 depletion decreased the cells under mitosis, but conversely increased the proportion of DNA synthesizing cells, thereby inhibited mitotic transition. The cell-cycle synchronization analysis and promoter analysis using live-cell imaging clarified that Meox1 oscillated throughout the cell-cycle and specifically emerged in G2/M phase. Finally, we revealed that Meox1 heterogenously expressed in the interstitial fibrotic are of human ventricular heart tissues from patients with end-stage heart failure. Notably, Meox1 expression was significantly correlated with the fibrosis-related genes in diseased ventricular heart tissues (n=15), suggesting the pathological relevance in clinical settings. Conclusion: Our findings identify a novel cell-cycle regulator and propose that Meox1 is a potential target for therapies aimed at preventing tissue fibrosis.

Blocking sirtuin 1 and 2 inhibits renal interstitial fibroblast activation and attenuates renal interstitial fibrosis in obstructive nephropathy.

Our recent studies revealed that blocking class I/II histone deacetylases (HDACs) inhibits renal interstitial fibroblast activation and proliferation and alleviates development of renal fibrosis. However, the effect of class III HDAC, particularly sirtuin 1 and 2 (SIRT1 and SIRT2), inhibition on renal fibrogenesis remains elusive. Here, we demonstrate that both SIRT1 and SIRT2 were expressed in cultured renal interstitial fibroblasts (NRK-49F). Exposure of NRK-49F to sirtinol, a selective inhibitor of SIRT1/2, or EX527 (6-chloro-2,3,4,9-tetrahydro-1H-carbazole-1-carboxamide), an inhibitor for SIRT1, resulted in reduced expression of fibroblast activation markers (α-smooth muscle actin, fibronectin, and collagen I) as well as proliferation markers (proliferating cell nuclear antigen, cyclin D1, cyclin E) in dose- and time-dependent manners. Treatment with a SIRT2 inhibitor, AGK2 (2-cyano-3-[5-(2,5-dichlorophenyl)-2-furanyl]-N-5-quinolinyl-2-propenamide), also dose- and time-dependently inhibited renal fibroblast activation and, to a lesser extent, cell proliferation. Furthermore, silencing of either SIRT1 or SIRT2 by small interfering RNA exhibited similar inhibitory effects. In a mouse model of obstructive nephropathy, administration of sirtinol attenuated deposition of collagen fibrils as well as reduced expression of α-smooth muscle actin, collagen I, and fibronectin in the injured kidney. SIRT1/2 inhibition-mediated antifibrotic effects are associated with dephosphorylation of epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor-β (PDGFRβ), and signal transducer and activator of transcription 3. Thus, SIRT1/2 activity may contribute to renal fibroblast activation and proliferation as well as renal fibrogenesis through activation of at least EGFR and PDGFRβ signaling. Blocking SIRT1/2 activation may have therapeutic potential for the treatment of chronic kidney disease.

Sonic hedgehog is a novel tubule-derived growth factor for interstitial fibroblasts after kidney injury.

Tubular epithelium constitutes the majority of the renal parenchyma and is the primary target of various kidney injuries. However, how the injured tubules drive interstitial fibroblast activation and proliferation remains poorly understood. Here, we investigated the role of sonic hedgehog (Shh), a secreted extracellular signaling protein, in fibroblast proliferation. Shh was induced in renal tubular epithelia in animal models of CKD induced by ischemia/reperfusion injury (IRI), adriamycin, or renal mass ablation, and in renal tubules of kidney biopsy specimens from CKD patients with different etiologies. Using Gli1-CreER(T2) reporter mice, we identified interstitial fibroblasts as the principal targets of renal Shh signaling in vivo. In vitro, incubation with Shh promoted normal rat kidney fibroblast proliferation, which was assessed by cell counting, MTT assay, and BrdU incorporation assay, and stimulated the induction of numerous proliferation-related genes. However, Shh had no effect on the proliferation of renal tubular epithelial cells. In vivo, overexpression of Shh promoted fibroblast expansion and aggravated kidney fibrotic lesions after IRI. Correspondingly, blockade of Shh signaling by cyclopamine, a small molecule inhibitor of Smoothened, inhibited fibroblast proliferation, reduced myofibroblast accumulation, and attenuated renal fibrosis. These studies identify Shh as a novel, specific, and potent tubule-derived growth factor that promotes interstitial fibroblast proliferation and activation. Our data also suggest that blockade of Shh signaling is a plausible strategy for therapeutic intervention of renal fibrosis.

Curcumin Ameliorates Renal Fibrosis by Inhibiting Local Fibroblast Proliferation and Extracellular Matrix Deposition

Renal fibrosis is mainly characterized by activation and proliferation of interstitial fibroblasts and by excessive synthesis and accumulation of extracellular matrix (ECM) components, including fibronectin (FN) and collagen. This study investigated the effects of curcumin on proliferation of renal interstitial fibroblasts and their underlying mechanisms in vivo and in vitro. ECM components were visualized by Sirius red and immunohistochemistry staining and quantified by western blot analysis in mice with unilateral ureteral obstruction (UUO). Duplex staining for proliferating cell nuclear antigen and α-smooth muscle actin (α-SMA), as well as MTT and flow cytometry assays, were performed to measure fibroblast proliferation. Protein expression of phosphorylated Smad2/3 (p-Smad2/3) and peroxisome proliferator-activated receptor-γ (PPAR-γ) were assessed by western blotting. Curcumin treatment decreased the accumulation of type I collagen and FN in the kidney of animals with UUO. Activation of rat renal interstitial fibroblasts (NRK-49F) was induced by TGF-β1. Curcumin treatment inhibited fibroblast proliferation and the cell cycle was arrested in the G1 phase. Curcumin treatment upregulated the expression of PPAR-γ and downregulated the expression of p-Smad2/3. These results suggest that curcumin treatment ameliorates renal fibrosis by reducing fibroblast proliferation and ECM accumulation mediated by PPAR-γ and Smad-dependent TGF-β1 signaling.

Platelet-derived growth factor-A and sonic hedgehog signaling direct lung fibroblast precursors during alveolar septal formation

Alveolar septal formation is required to support the respiration of growing mammals; in humans effacement of the alveolar surface and impaired gas exchange are critical features of emphysema and pulmonary fibrosis. Platelet-derived growth factor-A (PDGF-A) and its receptor PDGF-receptor-α (PDGFRα) are required for secondary septal elongation in mice during postnatal days 4 through 12 and they regulate the proliferation and septal location of interstitial fibroblasts. We examined lung fibroblasts (LF) to learn whether PDGFRα expression distinguished a population of precursor cells, with enhanced proliferative and migratory capabilities. We identified a subpopulation of LF that expresses sonic hedgehog (Shh) and stem cell antigen-1 (Sca1). PDGF-A and Shh both increased cytokinesis and chemotaxis in vitro, but through different mechanisms. In primary LF cultures, Shh signaled exclusively through a noncanonical pathway involving generation of Rac1-GTP, whereas both the canonical and noncanonical pathways were used by the Mlg neonatal mouse LF cell line. LF preferentially oriented their primary cilia toward their anterior pole during migration. Furthermore, a larger proportion of PDGFRα-expressing LF, which are more abundant at the septal tips, bore primary cilia compared with other alveolar cells. In pulmonary emphysema, destroyed alveolar septa do not regenerate, in part because cells fail to assume a configuration that allows efficient gas exchange. Better understanding how LF are positioned during alveolar development could identify signaling pathways, which promote alveolar septal regeneration.