Inhibition of MLL1-menin interaction attenuates renal fibrosis in obstructive nephropathy.

Mixed lineage leukemia 1 (MLL1), a histone H3 lysine 4 (H3K4) methyltransferase, exerts its enzymatic activity by interacting with menin and other proteins. It is unclear whether inhibition of the MLL1-menin interaction influences epithelial-mesenchymal transition (EMT), renal fibroblast activation, and renal fibrosis. In this study, we investigated the effect of disrupting MLL1-menin interaction on those events and mechanisms involved in a murine model of renal fibrosis induced by unilateral ureteral obstruction (UUO), in cultured mouse proximal tubular cells and renal interstitial fibroblasts. Injury to the kidney increased the expression of MLL1 and menin and H3K4 monomethylation (H3K4me1); MLL1 and menin were expressed in renal epithelial cells and renal interstitial fibroblasts. Inhibition of the MLL1-menin interaction by MI-503 administration or siRNA-mediated silencing of MLL1 attenuated UUO-induced renal fibrosis, and reduced expression of α-smooth muscle actin (α-SMA) and fibronectin. These treatments also inhibited UUO-induced expression of transcription factors Snail and Twist and transforming growth factor ß1 (TGF-ß1) while expression of E-cadherin was preserved. Moreover, treatment with MI-503 and transfection with either MLL siRNA or menin siRNA inhibited TGF-ß1-induced upregulation of α-SMA, fibronectin and Snail, phosphorylation of Smad3 and AKT, and downregulation of E-cadherin in cultured renal epithelial cells. Finally, MI-503 was effective in abrogating serum or TGFß1-induced transformation of renal interstitial fibroblasts to myofibroblasts in vitro. Taken together, these results suggest that targeting disruption of the MLL1-menin interaction attenuates renal fibrosis through inhibition of partial EMT and renal fibroblast activation.

Identification of histone deacetylase 8 as a novel therapeutic target for renal fibrosis.

Histone deacetylases (HDACs) have been shown to alleviate renal fibrosis, however, the role of individual HDAC isoforms in this process is poorly understood. In this study, we examined the role of HDAC8 in the development of renal fibrosis and partial epithelial-mesenchymal transitions (EMT). In a murine model of renal fibrosis induced by unilateral ureteral obstruction (UUO), HDAC8 was primarily expressed in renal tubular epithelial cells and time-dependently upregulated. This occurred in parallel with the deacetylation of cortactin, a nonhistone substrate of HDAC8, and increased expression of three fibrotic markers: α-smooth muscle actin, collagen 1, and fibronectin. Administration of PCI34051, a highly selective inhibitor of HDAC8, restored acetylation of contactin and reduced expression of those proteins. PCI34051 treatment also reduced the number of renal tubular epithelial cells arrested at the G2/M phase of the cell cycle and suppressed phosphorylation of Smad3, STAT3, ß-catenin, and expression of Snail after ureteral obstruction. In contrast, HDAC8 inhibition reversed UUO-induced downregulation of BMP7 and Klotho, two renoprotective proteins. In cultured murine proximal tubular cells, treatment with PCI34051 or specific HDAC8 siRNA was also effective in inhibiting transforming growth factor ß1 (TGFß1)-induced deacetylation of contactin, EMT, phosphorylation of Smad3, STAT3, and ß-catenin, upregulation of Snail, and downregulation of BMP7 and Klotho. Collectively, these results suggest that HDAC8 activation is required for the EMT and renal fibrogenesis by activation of multiple profibrotic signaling and transcription factors, and suppression of antifibrotic proteins. Therefore, targeting HDAC8 may be novel therapeutic approach for treatment of renal fibrosis.

Treprostinil, a prostacyclin analog, ameliorates renal ischemia-reperfusion injury: preclinical studies in a rat model of acute kidney injury.

BACKGROUND: Renal ischemia-reperfusion injury (IRI) is a major factor causing acute kidney injury (AKI). No pharmacological treatments for prevention or amelioration of I/R-induced renal injury are available. Here we investigate the protective effects of treprostinil, a prostacyclin analog, against renal IRI in vivo. METHODS: Male Sprague Dawley rats were subjected to bilateral renal ischemia (45 min) followed by reperfusion for 1-168 h. Treprostinil (100 ng/kg/min) or placebo was administered subcutaneously for 18-24 h before ischemia. RESULTS: Treatment with treprostinil both significantly reduced peak elevation and accelerated the return to baseline levels for serum creatinine and blood urea nitrogen versus I/R-placebo animals following IRI. I/R-treprostinil animals exhibited reduced histopathological features of tubular epithelial injury versus I/R-placebo animals. IRI resulted in a marked induction of messenger RNA coding for kidney injury biomarkers, kidney injury molecule-1 and neutrophil gelatinase-associated lipocalin and for pro-inflammatory cytokines chemokine (C-C motif) ligand 2, interleukin 1ß, interleukin 6 and intracellular adhesion molecular 1 in animals treated with placebo only relative to sham controls. Upregulation of expression of all these genes was significantly suppressed by treprostinil. Treprostinil significantly suppressed the elevation in renal lipid peroxidation found in the I/R-placebo group at 1-h post-reperfusion. In addition, renal protein expression of cleaved poly(ADP-ribose) polymerase 1 and caspase-3, -8 and -9 in I/R-placebo animals was significantly inhibited by treprostinil. CONCLUSIONS: This study demonstrates the efficacy of treprostinil in ameliorating I/R-induced AKI in rats by significantly improving renal function early post-reperfusion and by inhibiting renal inflammation and tubular epithelial apoptosis. Importantly, these data suggest that treprostinil has the potential to serve as a therapeutic agent to protect the kidney against IRI in vivo.

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-ß1 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.

Targeting histone methyltransferase enhancer of zeste homolog-2 inhibits renal epithelial-mesenchymal transition and attenuates renal fibrosis.

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. Its role in renal epithelial-mesenchymal transition (EMT) remains unknown. In this study, we found that EZH2 and H3K27me3 were highly expressed in mouse kidney with unilateral ureteral obstruction and cultured mouse kidney proximal tubular (TKPT) cells undergoing EMT. Inhibition of EZH2 with 3-deazaneplanocin A (3-DZNeP) attenuated renal fibrosis, which was associated with preserving E-cadherin expression and inhibiting Vimentin up-regulation in the obstructed kidney. Treatment with 3-DZNeP or transfection of EZH2 siRNA also inhibited TGF-ß1-induced EMT of TKPT cells. Injury to the kidney or cultured TKPT cells resulted in up-regulation of Snail-l family transcriptional repressor (Snail)-1 and Twist family basic helix-loop-helix (BHLH) transcription factor (Twist)-1, which are 2 transcription factors, and down-regulation of phosphatase and tensin homolog, a protein tyrosine phosphatase associated with inhibition of PI3K-protein kinase B (AKT) signaling; EZH2 inhibition or silencing reversed all those responses. 3-DZNeP was also effective in suppressing epithelial arrest at the G2/M phase and dephosphorylating AKT and ß-catenin in vivo and in vitro. These data indicate that EZH2 activation contributes to renal EMT and fibrosis through activation of multiple signaling pathways and suggest that EZH2 has potential as a therapeutic target for treatment of renal fibrosis.-Zhou, X., Xiong, C., Tolbert, E., Zhao, T. C., Bayliss, G., Zhuang, S. Targeting histone methyltransferase enhancer of zeste homolog-2 inhibits renal epithelial-mesenchymal transition and attenuates 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 TGFß1-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 TGFß receptor 1 expression, and 3-DZNeP administration prevented these effects. 3-DZNeP also suppressed phosphorylation of the renal EGF and PDGFß 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.

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-ß1 (TGF-ß1). 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-ß1 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 we suggest that Src is a potential therapeutic target for treatment of chronic renal fibrosis.

P2X7 receptor inhibition protects against ischemic acute kidney injury in mice.

Activation of the purinergic P2X7 receptor (P2X7R) has been associated with the development of experimental nephritis and diabetic and hypertensive nephropathy. However, its role in acute kidney injury (AKI) remains unknown. In this study, we examined the effects of P2X7R inhibition in a murine model of ischemia-reperfusion (I/R)-induced AKI using A438079, a selective inhibitor of P2X7R. At 24 h after I/R, mice developed renal dysfunction and renal tubular damage, which was accompanied by elevated expression of P2X7R. Early administration of A438079 immediately or 6 h after the onset of reperfusion protected against renal dysfunction and attenuated kidney damage whereas delayed administration of A438079 at 24 h after restoration of perfusion had no protective effects. The protective actions of A438079 were associated with inhibition of renal tubule injury and cell death and suppression of renal expression of monocyte chemotactic protein-1 and regulated upon expression normal T cell expressed and secreted (RANTES). Moreover, I/R injury led to an increase in phosphorylation (activation) of extracellular signal-regulated kinases 1/2 in the kidney; treatment with A438079 diminished this response. Collectively, these results indicate that early P2X7R inhibition is effective against renal tubule injury and proinflammatory response after I/R injury and suggest that targeting P2X7R may be a promising therapeutic strategy for treatment of AKI.

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 ß (PDGFRß). 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.

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-ß (PDGFRß), 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 PDGFRß signaling. Blocking SIRT1/2 activation may have therapeutic potential for the treatment of chronic kidney disease.

Sustained activation of EGFR triggers renal fibrogenesis after acute kidney injury.

Severe acute kidney injury (AKI) is frequently accompanied by maladaptive repair and renal fibrogenesis; however, the molecular mechanisms that mediate these acute and chronic consequences of AKI remain poorly understood. In this study, we examined the role of epidermal growth factor receptor (EGFR) in these processes using waved-2 (Wa-2) mice, which have reduced EGFR activity, and their wild-type (WT) littermates after renal ischemia. Renal EGFR phosphorylation was induced within 2 days after ischemia, increased over time, and remained elevated at 28 days in WT mice, but this was diminished in Wa-2 mice. At the early stage of postischemia (2 days), Wa-2 mice developed more severe acute renal tubular damage with less reparative responses as indicated by enhanced tubular cell apoptosis, and reduced dedifferentiation and proliferation as compared to WT animals. At the late stage of postischemia (28 days), Wa-2 mice exhibited a less severe renal interstitial fibrosis as shown by reduced activation/proliferation of renal myofibroblasts and decreased deposition of extracellular matrix proteins. EGFR activation also contributed to cell cycle arrest at the G2/M phase, a cellular event associated with production of profibrogenetic factors, in the injured kidney. Collectively, these results indicate that severe AKI results in sustained activation of EGFR, which is required for reparative response of renal tubular cells initially, but eventually leads to fibrogenesis.

Genetic or pharmacologic blockade of EGFR inhibits renal fibrosis.

Although enhanced activation of the EGF receptor (EGFR) associates with the development and progression of renal fibrosis, the mechanisms linking these observations are not completely understood. Here, after unilateral ureteral obstruction (UUO), wild-type mice exhibited sustained EGFR phosphorylation in the kidney and developed renal fibrosis that was more severe than the renal fibrosis observed in waved-2 mice, which have reduced EGFR tyrosine kinase activity. Waved-2 mice also showed fewer renal tubular cells arrested at G2/M, reduced expression of α-smooth muscle actin (α-SMA), downregulation of multiple genes encoding profibrogenic cytokines, including TGF-ß1, and dephosphorylation of Smad3, STAT3, and ERK1/2. Administration of the specific EGFR inhibitor gefitinib recapitulated this phenotype in wild-type mice after UUO. Furthermore, inactivation of either EGFR or STAT3 reduced UUO-induced expression of lipocalin-2, a molecule associated with the pathogenesis of CKD. In cultured renal interstitial fibroblasts, inhibition of EGFR also abrogated TGF-ß1- or serum-induced phosphorylation of EGFR, STAT3, ERK1/2, and Smad3 as well as expression of α-SMA and extracelluar matrix proteins. Taken together, these data suggest that EGFR may mediate renal fibrogenesis by promoting transition of renal epithelial cells to a profibrotic phenotype, increased production of inflammatory factors, and activation of renal interstitial fibroblasts. Inhibition of EGFR may have therapeutic potential for fibrotic kidney disease.

Suramin inhibits renal fibrosis in chronic kidney disease.

The activation of cytokine and growth factor receptors associates with the development and progression of renal fibrosis. Suramin is a compound that inhibits the interaction of several cytokines and growth factors with their receptors, but whether suramin inhibits the progression of renal fibrosis is unknown. Here, treatment of cultured renal interstitial fibroblasts with suramin inhibited their activation induced by TGF-ß1 and serum. In a mouse model of obstructive nephropathy, administration of a single dose of suramin immediately after ureteral obstruction abolished the expression of fibronectin, largely suppressed expression of α-SMA and type I collagen, and reduced the deposition of extracellular matrix proteins. Suramin also decreased the expression of multiple cytokines including TGF-ß1 and reduced the interstitial infiltration of leukocytes. Moreover, suramin decreased expression of the type II TGF-ß receptor, blocked phosphorylation of the EGF and PDGF receptors, and inactivated several signaling pathways associated with the progression of renal fibrosis. In a rat model of CKD, suramin abrogated proteinuria, limited the decline of renal function, and prevented glomerular and tubulointerstitial damage. Collectively, these findings indicate that suramin is a potent antifibrotic agent that may have therapeutic potential for patients with fibrotic kidney diseases.

Delayed administration of suramin attenuates the progression of renal fibrosis in obstructive nephropathy.

We recently showed that suramin treatment prevents the onset of renal fibrosis in a model of obstructive nephropathy induced by unilateral ureteral obstruction (UUO). In this study, we further assessed the effect of delayed administration of suramin on the progression of tubulointerstitial fibrosis. Mice were given a single dose of suramin at 20 mg/kg starting at day 3 of obstruction, and kidneys were harvested after an additional 7 or 14 days of obstruction. Suramin completely blocked further increase in expression of type I collagen and fibronectin and largely suppressed expression of α-smooth muscle actin (α-SMA) in both treatment groups. UUO injury induced phosphorylation of Smad-3, a key mediator of transforming growth factor-ß (TGF-ß) signaling, epidermal growth factor receptor, and platelet-derived growth factor receptor after 3 days and further increased at 10 days after UUO injury. When suramin was administered at 3 days after obstruction, phosphorylation of these molecules was not further increased in the obstructed kidney. Suramin treatment also inhibited activation of signal transducer and activator of transcription 3 and extracellular signal-regulated kinase 1 and 2, two signaling pathways associated with renal fibrogenesis. Furthermore, delayed application of suramin suppressed TGF-ß1-induced expression of α-SMA and fibronectin in cultured renal interstitial fibroblasts. These results indicate that administration of suramin is effective in attenuating the progression of renal fibrosis after injury and suggest the potential clinical application of suramin as an antifibrotic treatment in patients with chronic kidney disease.

Treprostinil alleviates hepatic mitochondrial injury during rat renal ischemia-reperfusion injury.

BACKGROUND: Renal ischemia-reperfusion injury (IRI) causes acute kidney injury as well as liver injury. Renal IRI depletes hepatic antioxidants, promotes hepatic inflammation and dysfunction through Tlr9 upregulation. There is no treatment available for liver injury during renal IRI. This study examines the hepatoprotective role of treprostinil, a prostacyclin analog, during renal IRI. METHODS: Male Sprague-Dawley rats were divided into four groups: control, sham, IRI-placebo, or IRI-treprostinil and subjected to bilateral ischemia (45 min) followed by reperfusion (1-72 h). Placebo or treprostinil (100 ng/kg/min) was administered subcutaneously via an osmotic minipump. RESULTS: Treprostinil significantly reduced peak serum creatinine, BUN, ALT and AST levels vs. IRI-placebo. Treprostinil also restored hepatic levels of superoxide dismutase, glutathione, catalase, and Gclc expression to baseline, while reducing lipid peroxidation vs. IRI-placebo. Additionally, treprostinil significantly reduced elevated hepatic Tlr9, Il-1ß, Ccl2, Vcam1, and Serpine1 mRNA expression. Renal IRI increased hepatic apoptosis which was inhibited by treprostinil through reduced cytochrome c and cleaved caspase-3 protein expression. Treprostinil enhanced hepatic ATP concentrations and mitochondrial DNA copy number and improved mitochondrial dynamics by restoring Pgc-1α expression and significantly upregulating Mfn1, Mfn2, and Sirt3 levels, while reducing Drp-1 protein vs. IRI-placebo. Non-targeted semi-quantitative proteomics showed improved oxidative stress indices and ATP subunits in the IRI-treprostinil group. CONCLUSIONS: Treprostinil improved hepatic function and antioxidant levels, while suppressing the inflammatory response and alleviating Tlr9-mediated apoptotic injury during renal IRI. Our study provides evidence of treprostinil's hepatoprotective effect, which supports the therapeutic potential of treprostinil in reducing hepatic injury during renal IRI.

Treprostinil reduces mitochondrial injury during rat renal ischemia-reperfusion injury.

BACKGROUND: Renal ischemia-reperfusion injury (IRI) is a major factor contributing to acute kidney injury and it is associated with a high morbidity and mortality if untreated. Renal IRI depletes cellular and tissue adenosine triphosphate (ATP), which compromises mitochondrial function, further exacerbating renal tubular injury. Currently, no treatment for IRI is available. This study investigates the protective role of treprostinil in improving mitochondria biogenesis and recovery during rat renal IRI. METHODS: Male Sprague Dawley rats were randomly assigned to groups: control, sham, IRI-placebo or IRI-treprostinil and subjected to 45 min of bilateral renal ischemia followed by 1-72 h reperfusion. Placebo or treprostinil (100 ng/kg/min) was administered subcutaneously via an osmotic minipump. RESULTS: Treprostinil significantly reduced peak elevated serum creatinine (SCr) levels and accelerated normalization relative to IRI-placebo (p < 0.0001). Treatment with treprostinil also inhibited IRI-mediated renal apoptosis, mitochondrial oxidative injury (p < 0.05), and the release of cytochrome c (p < 0.01) vs. IRI-placebo. In addition, treprostinil preserved renal mitochondrial DNA copy number (p < 0.0001) and renal ATP levels (p < 0.05) to nearly those of sham-operated animals. Non-targeted semi-quantitative proteomics showed reduced levels of ATP synthase subunits in the IRI-placebo group which were restored to sham levels by treprostinil treatment (p < 0.05). Furthermore, treprostinil reduced renal IRI-induced upregulated Drp1 and pErk protein levels, and restored Sirt3 and Pgc-1α levels to baseline (p < 0.05). CONCLUSIONS: Treprostinil reduces mitochondrial-mediated renal apoptosis, inhibits mitochondria fission, and promotes mitochondria fusion, thereby accelerating mitochondrial recovery and protecting renal proximal tubules from renal IRI. These results support the clinical investigation of treprostinil as a viable therapy to reduce renal IRI.

Direct fibrogenic effects of aldosterone on normotensive kidney: an effect modified by 11ß-HSD activity.

Aldosterone (Aldo) can be a profibrotic factor in cardiovascular and renal tissues. This study tests the hypothesis that prolonged Aldo exposure is able to directly induce fibrotic changes in the kidney of a normal nonhypertensive animal. Immortalized rat proximal tubule cells (IRPTC) containing 11ß-hydroxysteroid dehydrogenase (11ß-HSD1) but no mineralocorticoid receptors (MR) and mouse inner medullary collecting duct cells (IMCD) containing 11ß-HSD2 and MR were examined. IRPTC exposed to Aldo or corticosterone (10 nM) for 48 h demonstrated no change in collagen production as assessed by Sirius red staining. In contrast, IMCD treated with Aldo exhibited a marked increase in the expression of collagen, fibronectin, and connective tissue growth factor (CTGF), whereas corticosterone alone had no effect. The Aldo-induced overexperession of collagen, fibronectin, and CTGF was substantially attenuated by the MR antagonist RU-318 and by the 11ß-HSD end product 11-dehydrocorticosterone, but not by the glucocorticoid receptor antagonist RU-486. In vivo, early fibrotic changes with elevated collagen, fibronectin, and CTGF expression were observed in kidneys isolated from normotensive adrenalectomized mice receiving a continuous infusion of Aldo (8 µg·kg(-1)·day(-1)) for 1 wk. These changes were not present in corticosterone-treated mice. Aldo-induced changes were attenuated in adrenally intact mice and in mice treated with RU-318 or 11-dehydrocorticosterone. Thus, extended Aldo exposure produces fibrotic changes in cells containing MR and in normal kidneys. MR antagonists and the end products of 11ß-HSD attenuate these fibrogenic effects.

A novel STAT3 inhibitor, S3I-201, attenuates renal interstitial fibroblast activation and interstitial fibrosis in obstructive nephropathy.

Accumulation of both interstitial myofibroblasts and excessive production of extracellular matrix proteins is a common pathway contributing to chronic kidney disease. In a number of tissues, activation of STAT3 (signal transducer and activator of transcription 3) increases expression of multiple profibrotic genes. Here, we examined the effect of a STAT3 inhibitor, S3I-201, on activation of renal interstitial fibroblasts and progression of renal fibrosis. Treatment of cultured rat renal interstitial fibroblasts with S3I-201 inhibited their activation, as evidenced by dose- and time-dependent blockade of alpha-smooth muscle actin and fibronectin expression. In a mouse model of renal interstitial fibrosis induced by unilateral ureteral obstruction, STAT3 was activated, and administration of S3I-201 attenuated both this activation and extracellular matrix protein deposition following injury. S3I-201 reduced infiltration of the injured kidney by inflammatory cells and suppressed the injury-induced expression of fibronectin, alpha-smooth muscle actin, and collagen type-1 proteins, as well as the expression of multiple cytokines. Furthermore, S3I-201 inhibited proliferation and induced apoptosis preferentially in renal interstitial fibroblasts of the obstructed kidney. Thus, our results suggest that increased STAT3 activity mediates activation of renal interstitial fibroblasts and the progression of renal fibrosis. Inhibition of STAT3 signaling with S3I-201 may hold therapeutic potential for fibrotic kidney diseases.

Inhibition of histone deacetylase activity attenuates renal fibroblast activation and interstitial fibrosis in obstructive nephropathy.

Activation of renal interstitial fibroblasts is critically involved in the development of tubulointerstitial fibrosis in chronic kidney diseases. In this study, we investigated the effect of trichostatin A (TSA), a specific histone deacetylase (HDAC) inhibitor, on the activation of renal interstitial fibroblasts in a rat renal interstitial fibroblast line (NRK-49F) and the development of renal fibrosis in a murine model of unilateral ureteral obstruction (UUO). alpha-Smooth muscle actin (alpha-SMA) and fibronectin, two hallmarks of fibroblast activation, were highly expressed in cultured NRK-49F cells, and their expression was inhibited in the presence of TSA. Similarly, administration of TSA suppressed the expression of alpha-SMA and fibronectin and attenuated the accumulation of renal interstitial fibroblasts in the kidney after the obstructive injury. Activation of renal interstitial fibroblasts was accompanied by phosphorylation of signal transducer and activator of transcription 3 (STAT3), and TSA treatment also abolished these responses. Furthermore, inhibition of the STAT3 pathway with AG490 inhibited expression of alpha-SMA and fibronectin in NRK-49F cells. Finally, TSA treatment inhibited tubular cell apoptosis and caspase-3 activation in the obstructive kidney. Collectively, we suggest that pharmacological HDAC inhibition may induce antifibrotic activity by inactivation of renal interstitial fibroblasts and inhibition of renal tubular cell death. STAT3 may mediate those actions of HDACs.

Pharmacological targeting of BET proteins inhibits renal fibroblast activation and alleviates renal fibrosis.

Bromodomain and extra-terminal (BET) protein inhibitors have been shown to effectively inhibit tumorgenesis and ameliorate pulmonary fibrosis by targeting bromodomain proteins that bind acetylated chromatin markers. However, their pharmacological effects in renal fibrosis remain unclear. In this study, we examined the effect of I-BET151, a selective and potent BET inhibitor, on renal fibroblast activation and renal fibrosis. In cultured renal interstitial fibroblasts, exposure of cells to I-BET151, or silencing of bromodoma in-containing protein 4 (Brd4), a key BET protein isoform, significantly reduced their activation as indicated by decreased expression of α-smooth muscle actin, collagen 1 and fibronectin. In a murine model of renal fibrosis induced by unilateral ureteral obstruction (UUO), administration of I-BET151 suppressed the deposition of extracellular matrix proteins, renal fibroblast activation and macrophage infiltration. Mechanistically, I-BET151 treatment abrogated UUO-induced phosphorylation of epidermal growth factor receptor and platelet growth factor receptor-ß. It also inhibited the activation of Smad-3, STAT3 and NF-κB pathways, as well as the expression of c-Myc and P53 transcription factors in the kidney. Moreover, BET inhibition resulted in the reduction of renal epithelial cells arrested at the G2/M phase of cell cycle after UUO injury. Finally, injury to the kidney up-regulated Brd4, and I-BET151 treatment abrogated its expression. Brd4 was also highly expressed in human fibrotic kidneys. These data indicate that BET proteins are implicated in the regulation of signaling pathways and transcription factors associated with renal fibrogenesis, and suggest that pharmacological inhibition of BET proteins could be a potential treatment for renal fibrosis.