Activation Of Renal Interstitial Fibroblasts Research Articles

Pharmacological inhibition of Src family kinases attenuates hyperuricemic nephropathy.

Hyperuricemia is an independent risk factor for chronic kidney disease and contributes to renal fibrosis. This study aims to investigate the effect of Src family kinase (SFK) inhibition on the development of hyperuricemic nephropathy (HN) and the mechanisms involved. In a rat model of HN, feeding rats a mixture of adenine and potassium oxonate increased Src phosphorylation, severe glomerular sclerosis, and renal interstitial fibrosis, accompanied by renal dysfunction and increased urine microalbumin excretion. Administration of PP1, a highly selective SFK inhibitor, prevented renal dysfunction, reduced urine microalbumin, and inhibited activation of renal interstitial fibroblasts and expression of extracellular proteins. PP1 treatment also inhibited hyperuricemia-induced activation of the TGF-β1/Smad3, STAT3, ERK1/2, and NF-κB signaling pathways and expression of multiple profibrogenic cytokines/chemokines in the kidney. Furthermore, PP1 treatment significantly reduced serum uric acid levels and xanthine oxidase activity. Thus, blocking Src can attenuate development of HN via a mechanism associated with the suppression of TGF-β1 signaling, inflammation, and uric acid production. The results suggest that Src inhibition might be a promising therapeutic strategy for HN.

Deletion of STAT3 from Foxd1 cell population protects mice from kidney fibrosis by inhibiting pericytes trans-differentiation and migration.

Signal transduction and activator of transcription 3 (STAT3) is a key transcription factor implicated in the pathogenesis of kidney fibrosis. Although Stat3 deletion in tubular epithelial cells is known to protect mice from fibrosis, vFoxd1 cells remains unclear. Using Foxd1-mediated Stat3 knockout mice, CRISPR, and inhibitors of STAT3, we investigate its function. STAT3 is phosphorylated in tubular epithelial cells in acute kidney injury, whereas it is expanded to interstitial cells in fibrosis in mice and humans. Foxd1-mediated deletion of Stat3 protects mice from folic-acid- and aristolochic-acid-induced kidney fibrosis. Mechanistically, STAT3 upregulates the inflammation and differentiates pericytes into myofibroblasts. STAT3 activation increases migration and profibrotic signaling in genome-edited, pericyte-like cells. Conversely, blocking Stat3 inhibits detachment, migration, and profibrotic signaling. Furthermore, STAT3 binds to the Collagen1a1 promoter in mouse kidneys and cells. Together, our study identifies a previously unknown function of STAT3 that promotes kidney fibrosis and has therapeutic value in fibrosis.

Pharmacologic Targeting of BET Proteins Attenuates Hyperuricemic Nephropathy in Rats.

Hyperuricemia is an independent risk factor for renal damage and promotes the progression of chronic kidney disease. In this study, we investigated the effect of I-BET151, a small-molecule inhibitor targeting the bromodomain and extraterminal (BET) proteins, on the development of hyperuricemic nephropathy (HN), and the mechanisms involved. Expression levels of bromodomain-containing protein 2 and 4, but not 3 were increased in the kidney of rats with HN; administration of I-BET151 effectively prevented renal dysfunction, decreased urine microalbumin, and attenuated renal fibrosis as indicated by reduced activation of renal interstitial fibroblasts and expression of fibronectin and collagen I in HN rats. Mechanistic studies show that I-BET151 treatment inhibited transition of renal epithelial cells to a mesenchymal cell type as evidenced by preservation of E-cadherin and reduction of vimentin expression. This was coincident with reduced expression of TGF-β1 and dephosphorylation of Smad3 and ERK1/2. I-BET151 was also effective in inhibiting phosphorylation of NF-κB, expression of multiple cytokines and chemokines, and infiltration of macrophages to the injured kidney. Although there were increased serum levels of uric acid and xanthine oxidase, an enzyme that catalyzes production of uric acid, and decreased expression of renal organic anion transporter 1 and 3 that promote urate excretion in the model of HN, and reduced expression levels of urine uric acid, I-BET151 treatment did not affect these responses. Collectively, our results indicate that I-BET151 alleviates HN by inhibiting epithelial to mesenchymal transition and inflammation in association with blockade of TGF-β, ERK1/2 and NF-κB signaling.

Effect of Shenkang on renal fibrosis and activation of renal interstitial fibroblasts through the JAK2/STAT3 pathway

BackgroundActivation of renal fibroblasts is a critical mechanism in the process of renal fibrosis. As a commonly used herbal formula, Shenkang (SK) has been found to attenuate renal fibrosis and renal parenchyma destruction. However, the effect of SK on renal fibroblast activation in unilateral ureteral obstruction (UUO) mice and its molecular mechanism remain undetermined. The present study was performed to elucidate the effect of SK on renal fibroblast activation and renal fibrosis, as well as the potential underlying mechanism, in both NRK-49F cells and UUO mice.MethodsNRK-49F cells were stimulated with 10 ng/ml TGF-β1 for 48 h. After SK treatment, the CCK-8 method was used to evaluate cell viability. Thirty-six C57BL/6 mice were randomly divided into the sham group, UUO group, angiotensin receptor blocker (ARB) group, and SK high-, moderate- and low-dose groups. UUO was induced in mice except those in the sham group. Drugs were administered 1 day later. On the 13th day, the fractional anisotropy (FA) value was determined by MRI to evaluate the degree of renal fibrosis. After 14 days, serum indexes were assessed. Hematoxylin and eosin (HE) and Sirius red staining were used to observe pathological morphology and the degree of fibrosis of the affected kidney. Western blotting and PCR were used to assess the expression of related molecules in both cells and animals at the protein and gene levels.ResultsOur results showed that SK reduced extracellular matrix (ECM) and α-smooth muscle actin (α-SMA) expression both in vitro and in vivo and attenuated renal fibrosis and the pathological lesion degree after UUO, suppressing JAK2/STAT3 activation. Furthermore, we found that SK regulated the JAK2/STAT3 pathway regulators peroxiredoxin 5 (Prdx5) in vitro and suppressor of cytokine signaling protein 1 (SOCS1) and SOCS3 in vivo.ConclusionsThese results indicated that SK inhibited fibroblast activation by regulating the JAK2/STAT3 pathway, which may be a mechanism underlying its protective action in renal fibrosis.

Histone demethylase JMJD3 protects against renal fibrosis by suppressing TGFβ and Notch signaling and preserving PTEN expression.

Rationale: The Jumonji domain containing-3 (JMJD3), a specific histone demethylase for trimethylation on histone H3 lysine 27 (H3K27me3), is associated with the pathogenesis of many diseases, but its role in renal fibrosis remains unexplored. Here we examined the role of JMJD3 and mechanisms involved in the activation of renal fibroblasts and development of renal fibrosis.Methods: Murine models of 5/6 surgical nephrectomy (SNx) and ureteral unilateral obstruction (UUO) were used to assess the effect of a specific JMJD3 inhibitor, GSKJ4, and genetic deletion of JMJD3 from FOXD1 stroma-derived renal interstitial cells on the development of renal fibrosis and activation of renal interstitial fibroblasts. Cultured rat renal interstitial fibroblasts (NRK-49F) and mouse renal tubular epithelial cells (mTECs) were also used to examine JMJD3-mediated activation of profibrotic signaling.Results: JMJD3 and H3K27me3 expression levels were upregulated in the kidney of mice subjected to SNx 5/6 and UUO. Pharmacological inhibition of JMJD3 with GSKJ4 or genetic deletion of JMJD3 led to worsening of renal dysfunction as well as increased deposition of extracellular matrix proteins and activation of renal interstitial fibroblasts in the injured kidney. This was coincident with decreased expression of Smad7 and enhanced expression of H3K27me3, transforming growth factor β1 (TGFβ1), Smad3, Notch1, Notch3 and Jagged1. Inhibition of JMJD3 by GSK J4 or its specific siRNA also resulted in the similar responses in cultured NRK-49F and mTECs exposed to serum or TGFβ1. Moreover, JMJD3 inhibition augmented phosphorylation of AKT and ERK1/2 in vivo and in vitro.Conclusion: These results indicate that JMJD3 confers anti-fibrotic effects by limiting activation of multiple profibrotic signaling pathways and suggest that JMJD3 modulation may have therapeutic effects for chronic kidney disease.

Advances in understanding the role of adiponectin in renal fibrosis

Renal fibrosis is characterized by the proliferation of renal intrinsic cells, activation of renal interstitial fibroblasts and deposition of extracellular matrix (ECM), processes that lead to the progressive loss of renal function. Renal fibrosis is characterized by the proliferation of renal intrinsic cells, activation of renal interstitial fibroblasts, and septal fibrosis is recognized as a marker for the progression of chronic kidney disease, a condition that is associated with high morbidity and mortality and is a significant public health burden. Despite extensive studies, there are no effective treatments for renal fibrosis. Adiponectin (APN) is a protein mainly produced by adipocytes that has anti-inflammatory and anti-atherosclerotic effects, improves insulin resistance and provides other salutary effects. Recent studies found that APN can inhibit ECM deposition by inhibiting inflammation and oxidative stress, and by regulating the TGF-β, AMPK, MCP-1 and other signalling pathways. Many recent studies have examined the roles of these pathways in the pathogenesis of renal fibrosis. In this article, we review the pathogenic mechanism of APN in renal fibrosis and provide a theoretical basis for delaying and blocking renal fibrosis by alteration of APN activity.

SUN-100 Exosome-packaged sonic hedgehog in cardiomyocyte plays a central role in type 2 cardiorenal syndrome

Cardiorenal syndrome type 2 (CRS2) is characterized by chronic abnormalities in cardiac function causing progressive chronic kidney disease (CKD). However, the mediators connecting heart and kidney remains largely unknown. In this study, we provide evidence supporting that exosomes containing Shh generated by injured cardiomyocyte plays a vital role in mediating cardiorenal connection in mouse model of transverse aortic constriction (TAC). Four weeks after TAC, mice were randomized into three groups: 1) sham control; 2) TAC mice; 3) TAC mice injected daily with cyclopamine (CPN), a specific inhibitor of hedgehog signaling. At 8 weeks after TAC, all animals were sacrificed. Urine, blood, heart and kidney tissues were collected for analysis. Another set of mice were randomized into three groups after 4 weeks of TAC: 1) sham control mice injected with pcDNA3 plasmid; 2) TAC mice injected with pcDNA3 plasmid; 3) TAC mice injected with shRab27a plasmid, a small GTPases implicated in regulating exosome biogenesis and secretion. These mice were injected with pcDNA3 or shRab27a plasmids once a week for three weeks beginning at 4 weeks after TAC. At 8 weeks after TAC, all animals were sacrificed. Urine, blood, heart and kidney tissues were collected for analysis. Exosomes were isolated from TAC mice serum and CRS2 patients’ serum, and incubated with normal rat kidney interstitial fibroblast (NRK-49F) cells. At 8 weeks after TAC, cardiac hypertrophy and fibrosis were prominent, as evidenced by increased expression of β-myosin heavy chain, α-actin and fibronectin. Echocardiography also revealed an impaired cardiac function in TAC mice. These cardiac lesions were accompanied by an upregulation of Shh and increased production of exosomes in cardiac tissue. Electron microscopy confirmed abundant exosomes derived from the injured cardiomyocytes in the heart of TAC mice. Blockade of Shh signaling by CPN ameliorated cardiac injury and restored heart function. Moreover, TAC mice also developed kidney injury secondary to chronic heart failure, manifested by proteinuria, kidney fibrosis, podocyte injury, and renal upregulation of Shh. CPN mitigated all these lesions in the kidneys. Notably, serum derived from TAC mice or CRS patients was able to induce renal interstitial fibroblast activation in vitro, indicating the circulating factors as mediators of cardiorenal connection. Furthermore, exosomes isolated from TAC serum was sufficient to cause fibroblast activation and matrix production. Furthermore, blockade of exosome biogenesis and secretion by knockdown of Rab27a expression not only protected against cardiac hypertrophy and fibrosis but also ameliorated renal fibrotic lesions. These studies demonstrate that heart-derived, circulating exosomes are a unique and effective vehicle to deliver Shh into the kidney after TAC. Targeting exosome generation and secretion or inhibition of Shh signaling could be novel therapeutic strategies for protecting both heart and kidney in cardiorenal syndrome.

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.

Blocking the histone lysine 79 methyltransferase DOT1L alleviates renal fibrosis through inhibition of renal fibroblast activation and epithelial-mesenchymal transition.

Disruptor of telomeric silencing-1 like (DOT1L) protein specifically catalyzes the methylation of histone H3 on Lys79 (H3K79) and is implicated in tumors. But its role in tissue fibrosis remains unclear. Here we demonstrated that injury to the kidney increased DOT1L expression and H3K79 dimethylation in renal tubular epithelial cells and myofibroblasts in a murine model of unilateral ureteral obstruction. Administration of EPZ5676, a highly selective inhibitor of DOT1L, attenuated renal fibrosis. Treatment with EPZ5676 or DOT1L small interfering RNA also inhibited TGF-β1 and serum-induced activation of renal interstitial fibroblasts and epithelial-mesenchymal transition (EMT) in vitro. Moreover, blocking DOT1L abrogated injury-induced epithelial G2/M arrest; reduced expression of Snail, Twist, and Notch1; and inactivated several profibrotic signaling molecules in the injured kidney, including Smad3, epidermal growth factor receptor, platelet-derived growth factor receptor, signal transducer and activator of transcription 3, protein kinase B, and NF-κB. Conversely, DOT1L inhibition increased expression of phosphatase and tensin homolog, a protein associated with dephosphorylation of tyrosine kinase receptors, and prevented decline in levels of Klotho and Smad7, 2 renoprotective factors. Thus, our data indicate that targeting DOT1L attenuates renal fibrosis through inhibition of renal fibroblasts and EMT by suppressing activation of multiple profibrotic signaling pathways while retaining expression of renoprotective factors.-Liu, L., Zou, J., Guan, Y., Zhang, Y., Zhang, W., Zhou, X., Xiong, C., Tolbert, E., Zhao, T. C., Bayliss, G., Zhuang, S. Blocking the histone lysine 79 methyltransferase DOT1L alleviates renal fibrosis through inhibition of renal fibroblast activation and epithelial-mesenchymal transition.

SUN-210 THE HISTONE DEMETHYLASE JUMONJI DOMAIN-CONTAINING PROTEIN 3 ACTS AS AN EPIGENETIC SUPPRESSOR OF RENAL FIBROSIS

The histone demethylase Jumonji domain-containing protein 3 (JMJD3) is the enzyme that induces demethylation of histone H3 lysine 27 (H3K27) and its activation is involved in the tumor genesis. Although activation of H3K27 methyltransferase has been shown to be required for renal interstitial fibroblast activation and renal fibrogenesis, the role of JMJD3 in these processes remains elusive.

Nintedanib, a triple tyrosine kinase inhibitor, attenuates renal fibrosis in chronic kidney disease.

Nintedanib (BIBF1120) is a triple kinase inhibitor of platelet-derived growth factor receptor (PDGFR), fibroblast growth factor receptors (FGFR), vascular endothelial growth factor receptor (VEGFR), and Src family kinase, which has recently been approved by FDA to treat idiopathic pulmonary fibrosis. Whether it affects renal fibrosis remains unknown. Here, we demonstrated that administration of nintedanib immediately or 3 days after unilateral ureteral obstruction (UUO) injury and with folic acid (FA) injection attenuated renal fibrosis and inhibited activation of renal interstitial fibroblasts. Delayed administration of nintedanib also partially reversed established renal fibrosis. Treatment with nintedanib blocked UUO-induced phosphorylation of PDGFRβ, FGFR1, FGFR2, VEGFR2, and several Src family kinases including Src, Lck, Lyn as well as activation of signal transducer and activator of transcription-3 (STAT3), nuclear factor-κB (NF-κB), and Smad-3 in the kidney. Furthermore, nintedanib inhibited UUO-elicited renal proinflammatory cytokine expression and macrophage infiltration. These data indicate that nintedanib is a potent anti-fibrotic agent in the kidney and may hold therapeutic potential as a treatment of chronic fibrotic kidney disease.

Pharmacologic targeting ERK1/2 attenuates the development and progression of hyperuricemic nephropathy in rats.

The pathogenesis of hyperuricemia-induced chronic kidney disease is largely unknown. In this study, we investigated whether extracellular signal–regulated kinases1/2 (ERK1/2) would contribute to the development of hyperuricemic nephropathy (HN). In a rat model of HN induced by feeding mixture of adenine and potassium oxonate, increased ERK1/2 phosphorylation and severe glomerular sclerosis and renal interstitial fibrosis were evident, in parallel with diminished levels of renal function and increased urine microalbumin excretion. Administration of U0126, which is a selective inhibitor of the ERK1/2 pathway, improved renal function, decreased urine microalbumin and inhibited activation of renal interstitial fibroblasts as well as accumulation of extracellular proteins. U0126 also inhibited hyperuricemia-induced expression of multiple profibrogenic cytokines/chemokines and infiltration of macrophages in the kidney. Furthermore, U0126 treatment suppressed xanthine oxidase, which mediates uric acid production. It also reduced expression of the urate anion exchanger 1, which promotes reabsorption of uric acid, and preserved expression of organic anion transporters 1 and 3, which accelerate uric acid excretion in the kidney of hyperuricemic rats. Finally, U0126 inhibited phosphorylation of Smad3, a key mediator in transforming growth factor (TGF-β) signaling. In cultured renal interstitial fibroblasts, inhibition of ERK1/2 activation by siRNA suppressed uric acid-induced activation of renal interstitial fibroblasts. Collectively, pharmacologic targeting of ERK1/2 can alleviate HN by suppressing TGF-β signaling, reducing inflammation responses, and inhibiting the molecular processes associated with elevation of blood uric acid levels in the body. Thus, ERK1/2 inhibition may be a potential approach for the prevention and treatment of hyperuricemic nephropathy.

Effect of zinc deficiency on mouse renal interstitial fibrosis in diabetic nephropathy.

There is emerging evidence that tubulointerstitial fibrosis is the final common pathway of the majority of chronic progressive renal diseases, including diabetic nephropathy (DN). Zinc, an essential dietary element, has been suggested to be important for a number of protein functions during fibrosis invivo and invitro. However, the effect of zinc deficiency (ZnD) on renal interstitial fibrosis in DN remains unclear. The present study investigated the effect and the underlying mechanisms of ZnD on renal interstitial fibrosis during DN using an streptozotocin‑induced model of diabetes with immunofluorescence staining and western blot analysis. The present study identified that dietary zinc restriction significantly decreased zinc concentrations in the plasma and mouse kidney. ZnD enhanced albuminuria and extracellular matrix protein expression, associated with diabetic renal interstitial fibrosis by activation of renal interstitial fibroblasts and regulation of the expression of fibrosis‑associated factors, which may be mediated by the activation of fibroblasts via the TGF‑β/Smad signaling pathway. The data indicates that ZnD serves an important role in the pathogenic mechanisms of renal interstitial fibrosis during the development of DN.

Src inhibition blocks renal interstitial fibroblast activation and ameliorates renal fibrosis.

Increased Src activity has been associated with the pathogenesis of renal tumors and some glomerular diseases, but its role in renal interstitial fibrosis remains elusive. To evaluate this, cultured renal interstitial fibroblasts (NRK-49F) were treated with PP1, a selective inhibitor of Src. This resulted in decreased expression of α-smooth muscle actin, fibronectin, and collagen I in response to serum, angiotension II, or transforming growth factor-β1 (TGF-β1). Silencing Src with siRNA also inhibited expression of those proteins. Furthermore, inhibition of Src activity blocked renal fibroblast proliferation. In a murine model of renal interstitial fibrosis induced by unilateral ureteral obstruction, the active form of Src (phopsho-Src Tyr416) was upregulated in both renal interstitial fibroblasts and renal tubular cells of the fibrotic kidney. Its inactivation reduced renal fibroblast activation and attenuated extracellular matrix protein deposition. Src inhibition also suppressed activation of TGF-β1 signaling, activation of the epidermal growth factor receptor and STAT3, and reduced the number of renal epithelial cells arrested at the G2/M phase of the cell cycle after ureteral obstruction. Thus, Src is an important mediator of renal interstitial fibroblast activation and renal fibrosis, and suggest that Src is a potential therapeutic target for treatment of chronic renal fibrosis.

Enhancer of Zeste Homolog 2 Inhibition Attenuates Renal Fibrosis by Maintaining Smad7 and Phosphatase and Tensin Homolog Expression.

Enhancer of zeste homolog 2 (EZH2) is a methyltransferase that induces histone H3 lysine 27 trimethylation (H3K27me3) and functions as an oncogenic factor in many cancer types. However, the role of EZH2 in renal fibrogenesis remains unexplored. In this study, we found high expression of EZH2 and H3K27me3 in cultured renal fibroblasts and fibrotic kidneys from mice with unilateral ureteral obstruction and humans with CKD. Pharmacologic inhibition of EZH2 with 3-deazaneplanocin A (3-DZNeP) or GSK126 or siRNA-mediated silencing of EZH2 inhibited serum- and TGFβ1-induced activation of renal interstitial fibroblasts in vitro, and 3-DZNeP administration abrogated deposition of extracellular matrix proteins and expression of α-smooth muscle actin in the obstructed kidney. Injury to the kidney enhanced Smad7 degradation, Smad3 phosphorylation, and TGFβ receptor 1 expression, and 3-DZNeP administration prevented these effects. 3-DZNeP also suppressed phosphorylation of the renal EGF and PDGFβ receptors and downstream signaling molecules signal transducer and activator of transcription 3 and extracellular signal-regulated kinase 1/2 after injury. Moreover, EZH2 inhibition increased the expression of phosphatase and tensin homolog (PTEN), a protein previously associated with dephosphorylation of tyrosine kinase receptors in the injured kidney and serum-stimulated renal interstitial fibroblasts. Finally, blocking PTEN with SF1670 largely diminished the inhibitory effect of 3-DZNeP on renal myofibroblast activation. These results uncovered the important role of EZH2 in mediating the development of renal fibrosis by downregulating expression of Smad7 and PTEN, thus activating profibrotic signaling pathways. Targeted inhibition of EZH2, therefore, could be a novel therapy for treating CKD.

Sustained Activation of Wnt/β-Catenin Signaling Drives AKI to CKD Progression.

AKI is increasingly recognized as a major risk factor for progression to CKD. However, the factors governing AKI to CKD progression are poorly understood. In this study, we investigated this issue using moderate (20 minutes) and severe (30 minutes) ischemia/reperfusion injury (IRI) in mice. Moderate IRI led to acute kidney failure and transient Wnt/β-catenin activation, which was followed by the restoration of kidney morphology and function. However, severe IRI resulted in sustained and exaggerated Wnt/β-catenin activation, which was accompanied by development of renal fibrotic lesions characterized by interstitial myofibroblast activation and excessive extracellular matrix deposition. To assess the role of sustained Wnt/β-catenin signaling in mediating AKI to CKD progression, we manipulated this signaling by overexpression of Wnt ligand or pharmacologic inhibition of β-catenin. In vivo, overexpression of Wnt1 at 5 days after IRI induced β-catenin activation and accelerated AKI to CKD progression. Conversely, blockade of Wnt/β-catenin by small molecule inhibitor ICG-001 at this point hindered AKI to CKD progression. In vitro, Wnt ligands induced renal interstitial fibroblast activation and promoted fibronectin expression. However, activated fibroblasts readily reverted to a quiescent phenotype after Wnt ligands were removed, suggesting that fibroblast activation requires persistent Wnt signaling. These results indicate that sustained, but not transient, activation of Wnt/β-catenin signaling has a decisive role in driving AKI to CKD progression.

Activation of Sirtuin-1 Promotes Renal Fibroblast Activation and Aggravates Renal Fibrogenesis.

Although activation of sirtuin-1 (SIRT1) has been shown to protect the kidney from acute injury, its role in renal fibrosis remains controversial since both inhibition and activation of SIRT1 have been reported to attenuate renal fibrosis. To resolve this conflict, we further examined the effect of SIRT1 activators on the activation of renal interstitial fibroblasts and development of renal fibrosis in vivo and in vitro. In a murine model of renal fibrosis induced by unilateral ureteral obstruction, administration of SRT1720 (N-[2-[3-(piperazin-1-ylmethyl)imidazo[2,1-b][1,3]thiazol-6-yl]phenyl]quinoxaline-2-carboxamide), a potent activator of SIRT1, accelerated deposition of collagen fibrils and increased expression of fibroblast activation markers (α-smooth muscle actin [α-SMA], collagen I, and fibronectin) in the obstructive kidney of mice. In cultured rat renal interstitial fibroblasts (NRK-49F), exposure of cells to SRT1720 or YK-3-237 (B-[2-methoxy-5-[(1E)-3-oxo-3-(3,4,5-trimethoxyphenyl)-1-propen-1-yl]phenyl]-boronic acid), another SIRT1 activator, also resulted in enhanced expression of α-SMA and fibronectin. Mechanistic studies showed that augmentation of renal fibrogenesis by SRT1720 is associated with elevated phosphorylation of epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor β (PDGFRβ). SRT1720 treatment also increased the phosphorylation of signal transducer and activator of transcription 3 and protein kinase B in the fibrotic kidney and NRK-49F cells. However, SRT1720 treatment did not affect expression of proliferating cell nuclear protein, a proliferation marker and activation of extracellular signal regulated kinase 1/2 in vitro and in vivo. These results indicate that SIRT1-activating compounds can provoke renal fibrogenesis through a mechanism involved in the activation of EGFR and PDGFR signaling pathways and suggest that long-term use of SIRT1 activators risks the development and progression of chronic kidney disease.

Paclitaxel attenuates renal interstitial fibroblast activation and interstitial fibrosis by inhibiting STAT3 signaling

Recent studies have demonstrated that paclitaxel might inhibit renal fibrosis. However, the underlying molecular mechanism remains unclear. In this study, we hypothesized that low-dose paclitaxel may block the STAT3 (signal transducer and activator of transcription 3) signaling to attenuate fibrosis in a mouse model with unilateral ureteral obstruction. Both NRK-49F cells and mice with unilateral ureteral obstruction were treated with paclitaxel. The results showed that paclitaxel treatment resulted in a dose- and time-dependent decrease in tyrosine-phosphorylated STAT3, and inhibited the expression of fibronectin, alpha-smooth muscle actin (α-SMA), and collagen I in cultured NRK-49F cells. S3I-201, an STAT3 inhibitor, also suppressed the expression of fibronectin, α-SMA, and collagen I in cultured NRK-49F cells. Mechanistically, paclitaxel treatment blocked the STAT3 activity by disrupting the association of STAT3 with tubulin and inhibiting STAT3 nucleus translocation. Furthermore, paclitaxel also ameliorated renal fibrosis by down-regulating the expression of fibronectin, α-SMA, and collagen I, and suppressed the infiltration of macrophages and production of TNF-α, IL-1β, TGF-β, and ICAM-1 (intercellular adhesion molecule 1) by inhibition of STAT3 activity in obstructive nephropathy. These results suggest that paclitaxel may block the STAT3 activity by disrupting the association of STAT3 with tubulin and inhibiting STAT3 nucleus translocation, consequently leading to the suppression of renal interstitial fibroblast activation and the development of renal fibrosis, and inhibition of proinflammatory cytokine production.

EGF Receptor Inhibition Alleviates Hyperuricemic Nephropathy.

Hyperuricemia is an independent risk factor for CKD and contributes to kidney fibrosis. In this study, we investigated the effect of EGF receptor (EGFR) inhibition on the development of hyperuricemic nephropathy (HN) and the mechanisms involved. In a rat model of HN induced by feeding a mixture of adenine and potassium oxonate, increased EGFR phosphorylation and severe glomerular sclerosis and renal interstitial fibrosis were evident, accompanied by renal dysfunction and increased urine microalbumin excretion. Administration of gefitinib, a highly selective EGFR inhibitor, prevented renal dysfunction, reduced urine microalbumin, and inhibited activation of renal interstitial fibroblasts and expression of extracellular proteins. Gefitinib treatment also inhibited hyperuricemia-induced activation of the TGF-β1 and NF-κB signaling pathways and expression of multiple profibrogenic cytokines/chemokines in the kidney. Furthermore, gefitinib treatment suppressed xanthine oxidase activity, which mediates uric acid production, and preserved expression of organic anion transporters 1 and 3, which promotes uric acid excretion in the kidney of hyperuricemic rats. Thus, blocking EGFR can attenuate development of HN via suppression of TGF-β1 signaling and inflammation and promotion of the molecular processes that reduce uric acid accumulation in the body.

Study on lead-induced activation of rat renal interstitial fibroblasts and the related mechanisms

Context: Lead is a common industrial toxicant and has been proved to be associated with the kidney damage.Objective: To investigate the effect and mechanism of lead on expression of rat renal interstitial fibroblast activation related protein.Materials and methods: The expression of activation related protein mRNA was measured by real-time PCR in the NRK/49F treated by lead acetate with different concentrations (0, 0.5, 1 and 2 µmol/L). The effects of lead acetate on the level of fibronectin (FN) and signal transduction factors (Smads protein) expression were observed by Western blot.Results: The mRNA expression of activation-related protein increased significantly after the cells were stimulated by lead acetate for 24 h. The lead acetate-treated group could upregulate the p-Smad2, p-Smad3 and FN protein expression compared with the control group. The level of Smad2/3 protein expression did not change in all groups, the expression of SnoN decreased significantly compared with the control group.Discussion and conclusion: Lead acetate could increase the mRNA expression of activation-related factors. It could promote inflammatory reaction induced by TGF-β via Smad signaling pathway. Lead acetate has the effect on inducing the renal fibrosis.