ABSTRACT
AIM: Serum levels of phospholipase A2 receptor antibody (PLA2R; SAb) and glomerular deposits of PLA2R antigen (GAg) have been detected in patients with idiopathic membranous nephropathy (IMN). However, the correlation between these immunologic factors and their associations with the status and prognosis of IMN remain uncertain. METHODS: Fifty-one patients with biopsy-proven IMN diagnosed between March of 2015 and December of 2016 were enrolled in this study. All the patients were followed until March of 2017.We used enzyme-linked immunosorbent assay and immunofluorescence to measure the SAb and GAg, respectively. RESULTS: The positive rate of GAg was significantly higher than SAb in patients with IMN (88.24 vs 66.77%, P = 0.017). Compared with SAb- patients, SAb+ patients had a higher baseline proteinuria (6.21 vs 3.40 g/24 h), lower serum albumin (22.49 ± 6.59 vs 29.09 ± 7.40 g/L) and poorer renal function (88.96 ± 21.17 vs 107.25 ± 20.04 mL/min per 1.73 m2 ), as well as a higher renal IgG4 level (P < 0.05). A comparison of SAb+/GAg+ and SAb-/GAg+ tissues yielded similar results (P < 0.01). Regarding prognosis, SAb- patients had a higher rate of complete remission after immunosuppressive treatment than SAb+ patients (P = 0.042). CONCLUSION: The disease status and prognosis correlated more closely with the SAb than with the GAg in our cohort of patients with IMN. Furthermore, SAb+ patients had more severe clinical symptoms and a worse prognosis, which was probably associated with increased IgG4 deposition.
Subject(s)
Glomerulonephritis, Membranous , Kidney Glomerulus , Receptors, Phospholipase A2/immunology , Autoantibodies/blood , Biomarkers/blood , Biopsy/methods , Correlation of Data , Enzyme-Linked Immunosorbent Assay , Female , Fluorescent Antibody Technique , Glomerulonephritis, Membranous/diagnosis , Glomerulonephritis, Membranous/immunology , Glomerulonephritis, Membranous/pathology , Glomerulonephritis, Membranous/therapy , Humans , Immunosuppressive Agents/therapeutic use , Kidney Glomerulus/immunology , Kidney Glomerulus/pathology , Male , Middle Aged , Prognosis , Retrospective StudiesABSTRACT
Obstructive nephropathy is the end result of a variety of diseases that block drainage from the kidney(s). Transforming growth factor-ß1 (TGF-ß1)/Smad3-driven renal fibrosis is the common pathogenesis of obstructive nephropathy. In this study, we identified petchiether A (petA), a novel small-molecule meroterpenoid from Ganoderma, as a potential inhibitor of TGF-ß1-induced Smad3 phosphorylation. The obstructive nephropathy was induced by unilateral ureteral obstruction (UUO) in mice. Mice received an intraperitoneal injection of petA/vehicle before and after UUO or sham operation. An in vivo study revealed that petA protected against renal inflammation and fibrosis by reducing the infiltration of macrophages, inhibiting the expression of proinflammatory cytokines (interleukin-1ß and tumour necrosis factor-α) and reducing extracellular matrix deposition (α-smooth muscle actin, collagen I and fibronectin) in the obstructed kidney of UUO mice; these changes were associated with suppression of Smad3 and NF-κB p65 phosphorylation. Petchiether A inhibited Smad3 phosphorylation in vitro and down-regulated the expression of the fibrotic marker collagen I in TGF-ß1-treated renal epithelial cells. Further, we found that petA dose-dependently suppressed Smad3-responsive promoter activity, indicating that petA inhibits gene expression downstream of the TGF-ß/Smad3 signalling pathway. In conclusion, our findings suggest that petA protects against renal inflammation and fibrosis by selectively inhibiting TGF-ß/Smad3 signalling.
Subject(s)
Kidney Diseases/drug therapy , Kidney Diseases/metabolism , NF-kappa B/metabolism , Signal Transduction , Smad3 Protein/metabolism , Terpenes/therapeutic use , Transforming Growth Factor beta/metabolism , Actins/metabolism , Animals , Cell Line , Collagen/metabolism , Fibronectins/metabolism , Fibrosis , Humans , Inflammation/pathology , Kidney/injuries , Kidney/pathology , Kidney Diseases/pathology , Male , Mice, Inbred C57BL , Phosphorylation , Terpenes/chemistry , Terpenes/pharmacology , Terpenes/toxicity , Ureteral Obstruction/drug therapy , Ureteral Obstruction/pathologyABSTRACT
BACKGROUND: Recent studies show that C-reactive protein (CRP) is not only a biomarker but also a pathogenic mediator contributing to the development of inflammation and ageing-related diseases. However, serum levels of CRP in the healthy ageing population remained unclear, which was investigated in the present study. METHODS: Serum levels of high sensitive C-reactive protein (hs-CRP), glucose (Glu), triglyceride (TG), cholesterol (CHOL), high-density lipoprotein cholesterol (HDL-c), low-density lipoprotein cholesterol (LDL-c), superoxide dismutase (SOD), serum creatinine (SCr), serum uric acid (SUA) were measured in 6060healthy subjects (3672 male and 2388 female, mean age:45.9 years) who received routine physical examination at Sun Yat-sen Memorial Hospital, Guangzhou, China. RESULTS: In total of 6060 healthy people, serum levels of hs-CRP were significantly increased with ageing (P < 0.05), particularly in those with age over 45-year-old (1.31[0.69-2.75] vs 1.05[0.53-2.16]mg/L, P < 0.001). Interestingly, levels of serum hs-CRP were significantly higher in male than female population (1.24[0.65-2.57] vs 1.07[0.53-2.29]mg/L, P < 0.001). Correlation analysis also revealed that serum levels of hs-CRP positively correlated with age and SUA, but inversely correlated with serum levels of HDL-c and SOD (all P < 0.05). CONCLUSIONS: Baseline levels of serum hs-CRP are increased with ageing and are significantly higher in male than female healthy population. In addition, elevated serum levels of hs-CRP are also associated with increased SUA but decreased HDL-c and SOD. Thus, serum levels of hs-CRP may be an indicator associated with ageing in healthy Chinese population.
ABSTRACT
Smad7 plays a protective role in chronic kidney disease; however, its role in acute kidney injury (AKI) remains unexplored. Here, we report that Smad7 protects against AKI by rescuing the G1 cell cycle arrest of tubular epithelial cells (TECs) in ischemia/reperfusion-induced AKI in mice in which Smad7 gene is disrupted or restored locally into the kidney. In Smad7 gene knockout (KO) mice, more severe renal impairment including higher levels of serum creatinine and massive tubular necrosis was developed at 48 h after AKI. In contrast, restored renal Smad7 gene locally into the kidney of Smad7 KO mice protected against AKI by promoting TEC proliferation identified by PCNA+ and BrdU+ cells. Mechanistic studies revealed that worsen AKI in Smad7 KO mice was associated with a marked activation of TGF-ß/Smad3-p21/p27 signaling and a loss of CDK2/cyclin E activities, thereby impairing TEC regeneration at the G1 cell cycle arrest. In contrast, restored Smad7 locally into the kidneys of Smad7 KO mice protected TECs from the G1 cell cycle arrest and promoted TEC G1/S transition via a CDK2/cyclin E-dependent mechanism. In conclusion, Smad7 plays a protective role in AKI. Blockade of TGF-ß/Smad3-p21/p27-induced G1 cell cycle arrest may be a key mechanism by which Smad7 treatment inhibits AKI. Thus, Smad7 may be a novel therapeutic agent for AKI.
Subject(s)
Acute Kidney Injury/metabolism , Epithelial Cells/metabolism , G1 Phase Cell Cycle Checkpoints , Smad7 Protein/metabolism , Acute Kidney Injury/genetics , Acute Kidney Injury/physiopathology , Animals , Epithelial Cells/cytology , Female , Humans , Kidney/cytology , Kidney/injuries , Kidney/metabolism , Male , Mice , Pregnancy , Signal Transduction , Smad7 Protein/genetics , Transforming Growth Factor beta/metabolismABSTRACT
Acute kidney injury (AKI) is exacerbated in C-reactive protein transgenic mice but alleviated in Smad3 knockout mice. Here we used C-reactive protein transgenic/Smad3 wild-type and C-reactive protein transgenic/Smad3 knockout mice to investigate the signaling mechanisms by which C-reactive protein promotes AKI. Serum creatinine was elevated, and the extent of tubular epithelial cell necrosis following ischemia/reperfusion-induced AKI was greater in C-reactive protein transgenics but was blunted when Smad3 was deleted. Exacerbation of AKI in C-reactive protein transgenics was associated with increased TGF-ß/Smad3 signaling and expression of the cyclin kinase inhibitor p27, but decreased phosphorylated CDK2 and expression of cyclin E. Concomitantly, tubular epithelial cell proliferation was arrested at the G1 phase in C-reactive protein transgenics with fewer cells entering the S-phase cell cycle as evidenced by fewer bromodeoxyuridine-positive cells. In contrast, the protection from AKI in C-reactive protein transgenic/Smad3 knockout mice was associated with decreased expression of p27 and promotion of CDK2/cyclin E-dependent G1/S transition of tubular epithelial cells. In vitro studies using tubular epithelial cells showed that C-reactive protein activates Smad3 via both TGF-ß-dependent and ERK/MAPK cross talk mechanisms, Smad3 bound directly to p27, and blockade of Smad3 or the Fc receptor CD32 prevented C-reactive protein-induced p27-dependent G1 cell cycle arrest. In vivo, treatment of C-reactive protein transgenics with a Smad3 inhibitor largely improved AKI outcomes. Thus, C-reactive protein may promote AKI by impairing tubular epithelial cell regeneration via the CD32-Smad3-p27-driven inhibition of the CDK2/cyclin E complex. Targeting Smad3 may offer a new treatment approach for AKI.
Subject(s)
Acute Kidney Injury/pathology , C-Reactive Protein/metabolism , Cyclin E/metabolism , Cyclin-Dependent Kinase 2/metabolism , Kidney Tubules/physiology , Smad3 Protein/metabolism , Acute Kidney Injury/blood , Animals , C-Reactive Protein/genetics , Cell Line, Tumor , Cell Proliferation , Creatinine/blood , Cyclin E/genetics , Cyclin-Dependent Kinase Inhibitor p27/metabolism , Disease Models, Animal , Epithelial Cells/metabolism , Epithelial Cells/physiology , G1 Phase Cell Cycle Checkpoints , Humans , Isoquinolines/pharmacology , Kidney Tubules/cytology , MAP Kinase Signaling System , Mice , Mice, Inbred C57BL , Mice, Knockout , Necrosis , Phosphorylation , Pyridines/pharmacology , Pyrroles/pharmacology , Rats , Receptors, IgG/metabolism , Regeneration , Smad3 Protein/antagonists & inhibitors , Smad3 Protein/genetics , Transforming Growth Factor beta/metabolismABSTRACT
Increasing evidence shows that microRNAs play an important role in kidney disease. However, functions of long noncoding RNAs (lncRNAs) in kidney diseases remain undefined. We have previously shown that TGF-ß1 plays a diverse role in renal inflammation and fibrosis and Smad3 is a key mediator in this process. In this study, we used RNA-sequencing to identify lncRNAs related to renal inflammation and fibrosis in obstructive nephropathy induced in Smad3 wild-type and knockout mice. We found that Arid2-IR was a Smad3-associated lncRNA as a Smad3 binding site was found in the promoter region of Arid2-IR and deletion of Smad3 abolished upregulation of Arid2-IR in the diseased kidney. In vitro knockdown of Arid2-IR from tubular epithelial cells produced no effect on TGF-ß-induced Smad3 signaling and fibrosis but inhibited interleukin-1ß-stimulated NF-κB-dependent inflammatory response. In contrast, overexpression of Arid2-IR promoted interleukin-1ß-induced NF-κB signaling and inflammatory cytokine expression without alteration of TGF-ß1-induced fibrotic response. Furthermore, treatment of obstructed kidney with Arid2-IR shRNA blunted NF-κB-driven renal inflammation without effect on TGF-ß/Smad3-mediated renal fibrosis. Thus, Arid2-IR is a novel lncRNA that functions to promote NF-κB-dependent renal inflammation. Blockade of Arid2-IR may represent a novel and specific therapy for renal inflammatory disease.
Subject(s)
Genetic Therapy/methods , Inflammation/therapy , Kidney Diseases/therapy , RNA, Long Noncoding/genetics , Transcription Factors/genetics , Animals , Cells, Cultured , Gene Knockdown Techniques , Interleukin-1beta/antagonists & inhibitors , Interleukin-1beta/genetics , Interleukin-1beta/metabolism , Kidney/metabolism , Kidney Diseases/genetics , Male , Mice , Mice, Inbred C57BL , NF-kappa B/antagonists & inhibitors , NF-kappa B/genetics , NF-kappa B/metabolism , Sequence Analysis, RNA , Signal Transduction , Smad3 Protein/genetics , Smad3 Protein/metabolism , Transcription Factors/metabolism , Transforming Growth Factor beta/genetics , Transforming Growth Factor beta/metabolismABSTRACT
We have previously shown that transforming growth factor-ß/Smad3-dependent miRNAs play a critical role in renal inflammation and fibrosis. However, off-target effects of miRNAs limit their therapeutic application. Recently, emerging roles of long noncoding RNAs (lncRNAs) in diseases have been recognized. In this study, we used high-throughput RNA sequencing to identify the Smad3-dependent lncRNAs related to renal inflammation and fibrosis in Smad3 knockout mouse models of unilateral ureteral obstructive nephropathy and immunologically induced anti-glomerular basement membrane glomerulonephritis. Compared with wild-type mice, 151 lncRNAs in the unilateral ureteral obstructive nephropathy kidney and 413 lncRNAs in kidneys with anti-glomerular basement membrane glomerulonephritis were significantly altered in Smad3 knockout mice. Among them, 21 common lncRNAs were up-regulated in wild-type, but down-regulated in Smad3 knockout, kidneys in both disease models in which progressive renal inflammation and fibrosis were abolished when the Smad3 gene was deleted or suppressed. Real-time PCR confirmed these findings and revealed the functional link between Smad3-dependent lncRNAs np_5318/np_17856 and progressive kidney injury. Results demonstrate that the identification and characterization of functional lncRNAs associated with kidney disease may represent a promising research direction into renal disorder and may lead to the development of new lncRNA therapies for kidney diseases.
Subject(s)
Inflammation/pathology , Kidney/metabolism , Kidney/pathology , RNA, Long Noncoding/metabolism , Sequence Analysis, RNA , Smad3 Protein/metabolism , Transforming Growth Factor beta/metabolism , Animals , Base Sequence , Binding Sites , Female , Fibrosis , Gene Expression Regulation , Gene Ontology , Glomerulonephritis/genetics , Glomerulonephritis/pathology , Inflammation/genetics , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Molecular Sequence Data , Protein Binding , RNA, Long Noncoding/genetics , Signal Transduction , Ureteral Obstruction/genetics , Ureteral Obstruction/pathologyABSTRACT
Inflammation and its consequent fibrosis are two main features of diabetic nephropathy (DN), but target therapy on these processes for DN remains yet ineffective. We report here that miR-29b is a novel therapeutic agent capable of inhibiting progressive renal inflammation and fibrosis in type 2 diabetes in db/db mice. Under diabetic conditions, miR-29b was largely downregulated in response to advanced glycation end (AGE) product, which was associated with upregulation of collagen matrix in mesangial cells via the transforming growth factor-ß (TGF-ß)/Smad3-dependent mechanism. These pathological changes were reversed by overexpressing miR-29b, but enhanced by knocking-down miR-29b. Similarly, loss of renal miR-29b was associated with progressive diabetic kidney injury, including microalbuminuria, renal fibrosis, and inflammation. Restored renal miR-29b by the ultrasound-based gene therapy was capable of attenuating diabetic kidney disease. Further studies revealed that inhibition of Sp1 expression, TGF-ß/Smad3-dependent renal fibrosis, NF-κB-driven renal inflammation, and T-bet/Th1-mediated immune response may be mechanisms associated with miR-29b treatment in db/db mice. In conclusion, miR-29b may play a protective role in diabetic kidney disease and may have therapeutic potential for diabetic kidney complication.
Subject(s)
Diabetes Mellitus, Type 2/therapy , Diabetic Nephropathies/genetics , Inflammation/therapy , MicroRNAs/therapeutic use , Animals , Diabetes Mellitus, Type 2/complications , Diabetes Mellitus, Type 2/genetics , Diabetic Nephropathies/pathology , Diabetic Nephropathies/therapy , Disease Models, Animal , Fibrosis/genetics , Fibrosis/therapy , Genetic Therapy , Humans , Inflammation/genetics , Inflammation/pathology , Mice , MicroRNAs/genetics , Signal Transduction , Smad3 Protein/genetics , Transforming Growth Factor beta/geneticsABSTRACT
CRP (C-reactive protein) is regarded as an inflammatory biomarker in AKI (acute kidney injury), but its exact role in AKI remains unclear. Thus we sought to investigate the role of CRP in AKI. Clinically, elevated serum CRP levels were found to associate closely with increased serum creatinine and urea levels (P<0.01) in patients with AKI, which then fell after recovery from AKI. To determine the role of CRP in AKI, an ischaemia/reperfusion mouse model of AKI was developed using Tg (transgenic) mice that express human CRP. Compared with the WT (wild-type) mice, CRP Tg mice developed more severe renal injury at 24 h after ischaemia as determined by significantly increased serum creatinine and tubular necrosis. This was associated with an impaired TEC (tubular epithelium cell) regeneration as shown by an over 60% reduction in PCNA+ (proliferating-cell nuclear antigen) and BrdU+ (bromodeoxyuridine) TECs in CRP Tg mice with AKI. In vitro, the addition of CRP to a human TEC line (HK-2) also largely suppressed the proliferation of TECs. The functional role of CRP in AKI was demonstrated further by the blocking of CRP binding to the FcγRII (Fcγ receptor II) with a neutralizing anti-CD32 antibody, which restored TEC proliferation and prevented AKI in CRP Tg mice. Moreover, we found that impaired G1/S transition by suppression of the phosphorylation of CDK2 (cyclin-dependent kinase 2) and expression of cyclin E may be a key mechanism by which CRP inhibits TEC regeneration during the AKI repair process. In conclusion, CRP plays a pathogenic role in AKI by inhibiting G1/S-dependent TEC regeneration. The results of the present study suggest that targeting CRP signalling may offer a new therapeutic potential for AKI.
Subject(s)
Acute Kidney Injury/metabolism , C-Reactive Protein/metabolism , Epithelial Cells/metabolism , G1 Phase Cell Cycle Checkpoints , Kidney Tubules/metabolism , Regeneration , Reperfusion Injury/metabolism , Acute Kidney Injury/etiology , Acute Kidney Injury/genetics , Acute Kidney Injury/pathology , Acute Kidney Injury/prevention & control , Adolescent , Adult , Aged , Animals , Antibodies, Neutralizing/pharmacology , Apoptosis , Biomarkers/blood , C-Reactive Protein/genetics , Cell Line , Cell Proliferation , Cyclin E/metabolism , Cyclin-Dependent Kinase 2/metabolism , Disease Models, Animal , Epithelial Cells/drug effects , Epithelial Cells/pathology , G1 Phase Cell Cycle Checkpoints/drug effects , Humans , Kidney Tubules/drug effects , Kidney Tubules/pathology , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Middle Aged , Phosphorylation , Receptors, IgG/antagonists & inhibitors , Receptors, IgG/metabolism , Regeneration/drug effects , Reperfusion Injury/etiology , Reperfusion Injury/genetics , Reperfusion Injury/pathology , Reperfusion Injury/prevention & control , Signal Transduction , Up-Regulation , Young AdultABSTRACT
The TGFß (transforming growth factor ß)/SMAD and NF-κB (nuclear factor κB) signalling pathways play a key role in hypertensive nephropathy. The present study examined whether targeting these pathways by SMAD7, a downstream inhibitor of both pathways, blocks AngII (angiotensin II)-induced hypertensive kidney disease in mice. A doxycycline-inducible SMAD7-expressing plasmid was delivered into the kidney by a non-invasive ultrasound-microbubble technique before and after AngII infusion. Results showed that pre-treatment with SMAD7 prevented AngII-induced progressive renal injury by inhibiting an increase in proteinuria and serum creatinine while improving the glomerular filtration rate. Similarly, treatment with SMAD7 in the established hypertensive nephropathy at day 14 after AngII infusion halted the progressive renal injury. These preventive and therapeutic effects of SMAD7 on hypertensive kidney injury were associated with inhibition of AngII-induced up-regulation of SMURF2 (SMAD-specific E3 ubiquitin protein ligase 2) and Sp1 (specificity protein 1), blockade of TGFß/Smad3-mediated renal fibrosis and suppression of NF-κB-driven renal inflammation. Moreover, overexpression of SMAD7 also prevented AngII-induced loss of renal miR-29b, an miRNA with an inhibitory role in both TGFß/Smad3 and NF-κB pathways. In conclusion, SMAD7 may be a therapeutic agent for AngII-mediated hypertensive nephropathy. Inhibition of the Sp1/SMAD3/NF-κB/miR-29b regulatory network may be a mechanism by which SMAD7 inhibits hypertensive nephropathy.
Subject(s)
Hypertension, Renal/therapy , Nephritis/therapy , Smad7 Protein/genetics , Angiotensin II , Animals , Disease Models, Animal , Gene Transfer Techniques , Genetic Therapy , Hypertension, Renal/chemically induced , Hypertension, Renal/genetics , Immunohistochemistry , Interleukin-1beta/metabolism , Kidney/drug effects , Kidney/pathology , Kidney Diseases/chemically induced , Kidney Diseases/pathology , Kidney Diseases/prevention & control , Macrophages/pathology , Male , Mice , Mice, Inbred Strains , NF-kappa B/metabolism , Nephritis/chemically induced , Nephritis/genetics , Real-Time Polymerase Chain Reaction , Signal Transduction , Tumor Necrosis Factor-alpha/metabolismABSTRACT
Blockade of transforming growth factor-ß (TGF-ß) signaling by Smad7 gene therapy is known to prevent experimental renal fibrosis. This study investigated whether Smad7 suppresses renal fibrosis via altering the renal expression of fibrosis-related microRNAs. Application of gene therapy into diseased kidneys of obstructive nephropathy and kidney cells by overexpressing Smad7 restored miR-29b but inhibited the expression of miR-192 and miR-21, resulting in blockade of renal fibrosis. Furthermore, Smad7 overexpression also suppressed advanced glycated end products- and angiotensin II-regulated expression of these microRNAs. In contrast, disruption of Smad7 gene in mice demonstrated opposite results by enhancing the loss of miR-29b and upregulation of miR-192 and miR-21, resulting in promotion of renal fibrosis in ligated kidneys of a model of obstructive nephropathy. More importantly, treatment with anti-miR-29b, miR-21 and miR-192 mimics in Smad7 overexpressing tubular epithelial cells abrogated the suppressive function of Smad7 on renal fibrosis, suggesting that these microRNAs act downstream of Smad7 to override the Smad7 function. In conclusion, Smad7 protects kidneys from fibrosis by regulating TGF-ß/Smad3-mediated renal expression of miR-21, miR-192, and miR-29b. Restored renal miR-29b but suppressed miR-192 and miR-21 may be a mechanism by which gene therapy with Smad7 inhibits renal fibrosis.
Subject(s)
Kidney Diseases/prevention & control , MicroRNAs/genetics , Smad7 Protein/genetics , Transforming Growth Factor beta1/genetics , Animals , Blotting, Western , Cell Line , Epithelial Cells/cytology , Epithelial Cells/metabolism , Fibrosis , Gene Expression Regulation , Gene Transfer Techniques , Genetic Therapy , Immunohistochemistry , In Situ Hybridization , Kidney Diseases/genetics , Kidney Diseases/pathology , Mice , Mice, Knockout , MicroRNAs/metabolism , Rats , Reverse Transcriptase Polymerase Chain Reaction , Smad7 Protein/metabolism , Transforming Growth Factor beta1/metabolismABSTRACT
The TGF-ß/Smad3 pathway plays a major role in tissue fibrosis, but the precise mechanisms are not fully understood. Here we identified microRNA miR-433 as an important component of TGF-ß/Smad3-driven renal fibrosis. The miR-433 was upregulated following unilateral ureteral obstruction, a model of aggressive renal fibrosis. In vitro, overexpression of miR-433 enhanced TGF-ß1-induced fibrosis, whereas knockdown of miR-433 suppressed this response. Furthermore, Smad3, but not Smad2, bound to the miR-433 promoter to induce its expression. Delivery of an miR-433 knockdown plasmid to the kidney by ultrasound microbubble-mediated gene transfer suppressed the induction and progression of fibrosis in the obstruction model. The antizyme inhibitor Azin1, an important regulator of polyamine synthesis, was identified as a target of miR-433. Overexpression of miR-433 suppressed Azin1 expression, while, in turn, Azin1 overexpression suppressed TGF-ß signaling and the fibrotic response. Thus, miR-433 is an important component of TGF-ß/Smad3-induced renal fibrosis through the induction of a positive feedback loop to amplify TGF-ß/Smad3 signaling, and may be a potential therapeutic target in tissue fibrosis.
Subject(s)
Carrier Proteins/metabolism , Kidney Diseases/metabolism , Kidney/metabolism , MicroRNAs/metabolism , Smad3 Protein/metabolism , Transforming Growth Factor beta1/metabolism , Animals , Anti-Glomerular Basement Membrane Disease/genetics , Anti-Glomerular Basement Membrane Disease/metabolism , Anti-Glomerular Basement Membrane Disease/pathology , Binding Sites , Carrier Proteins/genetics , Cell Line , Disease Models, Animal , Doxorubicin , Fibrosis , Kidney/pathology , Kidney Diseases/etiology , Kidney Diseases/genetics , Kidney Diseases/pathology , Kidney Diseases/prevention & control , Male , Mice , Mice, 129 Strain , Mice, Inbred C57BL , Mice, Knockout , MicroRNAs/genetics , Promoter Regions, Genetic , RNA Interference , Rats , Signal Transduction , Smad2 Protein/deficiency , Smad2 Protein/genetics , Smad2 Protein/metabolism , Smad3 Protein/deficiency , Smad3 Protein/genetics , Smad7 Protein/genetics , Smad7 Protein/metabolism , Time Factors , Transfection , Transforming Growth Factor beta1/genetics , Up-Regulation , Ureteral Obstruction/complicationsABSTRACT
TGF-ß (transforming growth factor-ß) and BMP-7 (bone morphogenetic protein-7), two key members in the TGF-ß superfamily, play important but diverse roles in CKDs (chronic kidney diseases). Both TGF-ß and BMP-7 share similar downstream Smad signalling pathways, but counter-regulate each other to maintain the balance of their biological activities. During renal injury in CKDs, this balance is significantly altered because TGF-ß signalling is up-regulated by inducing TGF-ß1 and activating Smad3, whereas BMP-7 and its downstream Smad1/5/8 are down-regulated. In the context of renal fibrosis, Smad3 is pathogenic, whereas Smad2 and Smad7 are renoprotective. However, this counter-balancing mechanism is also altered because TGF-ß1 induces Smurf2, a ubiquitin E3-ligase, to target Smad7 as well as Smad2 for degradation. Thus overexpression of renal Smad7 restores the balance of TGF-ß/Smad signalling and has therapeutic effect on CKDs. Recent studies also found that Smad3 mediated renal fibrosis by up-regulating miR-21 (where miR represents microRNA) and miR-192, but down-regulating miR-29 and miR-200 families. Therefore restoring miR-29/miR-200 or suppressing miR-21/miR-192 is able to treat progressive renal fibrosis. Furthermore, activation of TGF-ß/Smad signalling inhibits renal BMP-7 expression and BMP/Smad signalling. On the other hand, overexpression of renal BMP-7 is capable of inhibiting TGF-ß/Smad3 signalling and protects the kidney from TGF-ß-mediated renal injury. This counter-regulation not only expands our understanding of the causes of renal injury, but also suggests the therapeutic potential by targeting TGF-ß/Smad signalling or restoring BMP-7 in CKDs. Taken together, the current understanding of the distinct roles and mechanisms of TGF-ß and BMP-7 in CKDs implies that targeting the TGF-ß/Smad pathway or restoring BMP-7 signalling may represent novel and effective therapies for CKDs.
Subject(s)
Bone Morphogenetic Protein 7/metabolism , Renal Insufficiency, Chronic/metabolism , Smad Proteins/metabolism , Transforming Growth Factor beta/metabolism , Apoptosis/physiology , Biomarkers/metabolism , Bone Morphogenetic Protein 7/antagonists & inhibitors , Cell Proliferation , Fibrosis/metabolism , Humans , Renal Agents/therapeutic use , Renal Insufficiency, Chronic/drug therapy , Renal Insufficiency, Chronic/pathology , Renal Insufficiency, Chronic/physiopathology , Smad Proteins/antagonists & inhibitors , Transforming Growth Factor beta/antagonists & inhibitorsABSTRACT
Autoimmunity leads to the activation of innate effector pathways, proinflammatory cytokine production, and end-organ injury. Macrophage migration inhibitory factor (MIF) is an upstream activator of the innate response that mediates the recruitment and retention of monocytes via CD74 and associated chemokine receptors, and it has a role in the maintenance of B lymphocytes. High-expression MIF alleles also are associated with end-organ damage in different autoimmune diseases. We assessed the therapeutic efficacy of (S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester (ISO-1), an orally bioavailable MIF antagonist, in two distinct models of systemic lupus erythematosus: the NZB/NZW F1 and the MRL/lpr mouse strains. ISO-1, like anti-MIF, inhibited the interaction between MIF and its receptor, CD74, and in each model of disease, it reduced functional and histological indices of glomerulonephritis, CD74(+) and CXCR4(+) leukocyte recruitment, and proinflammatory cytokine and chemokine expression. Neither autoantibody production nor T and B cell activation were significantly affected, pointing to the specificity of MIF antagonism in reducing excessive proinflammatory responses. These data highlight the feasibility of targeting the MIF-MIF receptor interaction by small-molecule antagonism and support the therapeutic value of downregulating MIF-dependent pathways of tissue damage in systemic lupus erythematosus.
Subject(s)
Genetic Predisposition to Disease , Glomerulonephritis/prevention & control , Intramolecular Oxidoreductases/antagonists & inhibitors , Isoxazoles/therapeutic use , Lupus Erythematosus, Systemic/prevention & control , Macrophage Migration-Inhibitory Factors/antagonists & inhibitors , Amino Acid Sequence , Animals , Cell Migration Inhibition/drug effects , Cell Migration Inhibition/immunology , Female , Gene Expression Profiling , Glomerulonephritis/genetics , Glomerulonephritis/immunology , Humans , Intramolecular Oxidoreductases/biosynthesis , Isoxazoles/administration & dosage , Lupus Erythematosus, Systemic/genetics , Lupus Erythematosus, Systemic/immunology , Macrophage Migration-Inhibitory Factors/biosynthesis , Mice , Mice, Inbred MRL lpr , Mice, Inbred NZB , Mice, Knockout , Molecular Sequence Data , Random Allocation , Receptors, Immunologic/antagonists & inhibitors , Receptors, Immunologic/biosynthesisABSTRACT
Loss of microRNA-29 (miR-29) is known to be a mechanism of transforming growth factor-ß (TGF-ß)-mediated pulmonary fibrosis, but the therapeutic implication of miR-29 for pulmonary fibrosis remains unexplored. The present study investigated whether miR-29 had therapeutic potential for lung disease induced by bleomycin in mice. In addition, the signaling mechanisms that regulated miR-29 expression were investigated in vivo and in vitro. We found that miR-29 was a downstream target gene of Smad3 and negatively regulated by TGF-ß/Smad signaling in fibrosis. This was evidenced by the findings that mice or pulmonary fibroblasts null for Smad3 were protected against bleomycin or TGF-ß1-induced loss of miR-29 along with fibrosis in vivo and in vitro. Interestingly, overexpression of miR-29 could in turn negatively regulated TGF-ß and connective tissue growth factor (CTGF) expression and Smad3 signaling. Therefore, Sleeping Beauty (SB)-mediated miR-29 gene transfer into normal and diseased lung tissues was capable of preventing and treating pulmonary fibrosis including inflammatory macrophage infiltration induced by bleomycin in mice. In conclusion, miR-29 is negatively regulated by TGF-ß/Smad3 and has a therapeutic potential for pulmonary fibrosis. SB-mediated miR-29 gene therapy is a non-invasive therapeutic strategy for lung disease associated with fibrosis.
Subject(s)
MicroRNAs/metabolism , Pulmonary Fibrosis/genetics , Pulmonary Fibrosis/prevention & control , Animals , Bleomycin , Cells, Cultured , DNA Transposable Elements , Disease Models, Animal , Female , Fibroblasts/drug effects , Fibroblasts/metabolism , Gene Expression , Gene Expression Regulation/drug effects , Gene Transfer Techniques , Genetic Therapy , Mice , Mice, Inbred C57BL , Mice, Knockout , MicroRNAs/administration & dosage , Pulmonary Fibrosis/chemically induced , Signal Transduction , Smad3 Protein/genetics , Smad3 Protein/metabolism , Time Factors , Transforming Growth Factor beta/metabolism , Transforming Growth Factor beta/pharmacologyABSTRACT
AIM: Transforming growth factor-ß (TGF-ß) has been shown to play a role in peritoneal angiogenesis associated with peritoneal dialysis (PD). The present study investigated whether blockade of TGF-ß signalling with Smad7 has a therapeutic effect on PD induced-peritoneal angiogenesis. METHODS: A rat model of peritoneal dialysis was induced by a daily intraperitoneal injection of 4.25% Dianeal and lipopolysaccharides. PD rats were transfected with a doxycycline regulated, Smad7-expressing plasmid using an ultrasound-microbubble-mediated system on day 0 and day 14 after initiation of PD and an empty vector was used as control. Peritoneal microvessel density (MVD) in peritoneal tissue was assessed by anti-CD31 immunohistochemistry after 4 weeks of PD and peritoneal angiogenic growth factors, including vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and platelet-derived growth factor (PDGF) was also examined by immunofluorescence, western blot and reverse transcription-polymerase chain reaction. RESULTS: In contrast to the normal control group, at 4 weeks after PD, PD rats displayed peritoneal lesions, peritoneal angiogenesis and increased mRNA and protein expression of VEGF, bFGF and PDGF. Smad7 gene transfer significantly attenuated the peritoneal MVD and inhibited the upregulation of VEGF, bFGF and PDGF. Moreover, inhibition of peritoneal angiogenesis by overexpression of Smad7 was associated with inhibition of phosphorylation of Smad3 and downregulation of TGF-ß expression. CONCLUSION: Smad7 gene transfer via an ultrasound-microbubble-mediated system is able to attenuate peritoneal angiogenesis in a rat model of PD. Those results suggest that blockade of the TGF-ß/Smad signalling pathway may represent a novel therapeutic approach to prevent PD-induced peritoneal angiogenesis.
Subject(s)
Genetic Therapy/methods , Neovascularization, Pathologic/prevention & control , Peritoneal Cavity/blood supply , Peritoneal Dialysis/adverse effects , Smad7 Protein/biosynthesis , Transfection , Animals , Disease Models, Animal , Fibroblast Growth Factor 2/metabolism , Immunohistochemistry , Injections, Intraperitoneal , Male , Microbubbles , Neovascularization, Pathologic/genetics , Neovascularization, Pathologic/metabolism , Phospholipids/administration & dosage , Phosphorylation , Platelet Endothelial Cell Adhesion Molecule-1/metabolism , Platelet-Derived Growth Factor/metabolism , Rats , Rats, Sprague-Dawley , Smad3 Protein/metabolism , Smad7 Protein/genetics , Sulfur Hexafluoride/administration & dosage , Time Factors , Transforming Growth Factor beta/metabolism , Vascular Endothelial Growth Factor A/metabolismABSTRACT
Although Smad3 is a key mediator for fibrosis, its functional role and mechanisms in hypertensive nephropathy remain largely unclear. This was examined in the present study in a mouse model of hypertension induced in Smad3 knockout (KO) and wild-type (WT) mice by subcutaneous angiotensin II infusion and in vitro in mesangial cells lacking Smad3. After angiotensin II infusion, both Smad3 KO and WT mice developed equally high levels of blood pressure. However, disruption of Smad3 prevented angiotensin II-induced kidney injury by lowering albuminuria and serum creatinine (P < 0.01), inhibiting renal fibrosis such as collagen type I and IV, fibronectin, and α-SMA expression (all P < 0.01), and blocking renal inflammation including macrophage and T cell infiltration and upregulation of IL-1ß, TNF-α, and monocyte chemoattractant protein-1 in vivo and in vitro (all P < 0.001). Further studies revealed that blockade of angiotensin II-induced renal transforming growth factor (TGF)-ß1 expression and inhibition of Smurf2-mediated degradation of renal Smad7 are mechanisms by which Smad3 KO mice were protected from angiotensin II-induced renal fibrosis and NF-κB-driven renal inflammation in vivo and in vitro. In conclusion, Smad3 is a key mediator of hypertensive nephropathy. Smad3 promotes Smurf2-dependent ubiquitin degradation of renal Smad7, thereby enhancing angiotensin II-induced TGF-ß/Smad3-mediated renal fibrosis and NF-κB-driven renal inflammation. Results from this study suggest that inhibition of Smad3 or overexpression of Smad7 may be a novel therapeutic strategy for hypertensive nephropathy.
Subject(s)
Angiotensin II/pharmacology , Hypertension/complications , Kidney Diseases/etiology , Smad3 Protein/metabolism , Actins/biosynthesis , Albuminuria/physiopathology , Animals , Chemokine CCL2/biosynthesis , Collagen Type I/biosynthesis , Collagen Type IV/biosynthesis , Creatinine/blood , Female , Fibronectins/biosynthesis , Fibrosis , Hypertension/metabolism , Hypertension/physiopathology , Kidney Diseases/metabolism , Kidney Diseases/pathology , Kidney Diseases/physiopathology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , NF-kappa B/metabolism , Smad3 Protein/genetics , Smad3 Protein/physiology , Smad7 Protein/metabolism , Smad7 Protein/physiology , Transforming Growth Factor beta/metabolism , Transforming Growth Factor beta/physiology , Ubiquitin-Protein Ligases/metabolismABSTRACT
The role of X chromosome-linked inhibitor of apoptosis protein (XIAP)-associated factor 1 (XAF1) in mediating apoptosis has been reported but the underlying mechanism remains unclear. The present study was designed to examine the putative interaction between XAF1 and p53 and the functional importance of this interaction in regulation of apoptosis in human gastric and colon cancer cells. We first identified XAF1 as a novel target gene of p53 by the chromatin immunoprecipitation (CHIP) assay and demonstrated that wild-type p53, but not mutant p53, down-regulated XAF1 at both mRNA and protein levels, which acted mostly under the condition of high expression of XAF1 and was associated with the physical interaction between p53 and the XAF1 promoter. We also found that the over-expression of XAF1 led to activation of wild-type p53 via post-translational modification in cells with or without DNA damage, which resulting in p53 nuclear accumulation and its increased transcriptional activity and enhancing p53-dependent apoptosis. These findings suggest that a potential novel feedback loop exists between XAF1 and wild-type p53.
Subject(s)
Apoptosis/genetics , Colonic Neoplasms/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Neoplasm Proteins/metabolism , Stomach Neoplasms/metabolism , Tumor Suppressor Protein p53/metabolism , Adaptor Proteins, Signal Transducing , Apoptosis Regulatory Proteins , Cell Line, Tumor , Colonic Neoplasms/genetics , Gene Expression Regulation, Neoplastic , Humans , Intracellular Signaling Peptides and Proteins/genetics , Neoplasm Proteins/genetics , Promoter Regions, Genetic , Protein Processing, Post-Translational/genetics , Stomach Neoplasms/genetics , Tumor Suppressor Protein p53/geneticsABSTRACT
The main function of chemokines is to guide inflammatory cells in their migration to sites of inflammation. During the last 2 decades, an expanding number of chemokines and their receptors have driven broad inquiry into how inflammatory cells are recruited in a variety of diseases. Although this review focuses on chemokines and their receptors in renal injury, proinflammatory IL-17, TGFß, and TWEAK signaling pathways also play a critical role in their expression. Recent studies in transgenic mice as well as blockade of chemokine signaling by neutralizing ligands or receptor antagonists now allow direct interrogation of chemokine action. The emerging role of regulatory T cells and Th17 cells during renal injury also forges tight relationships between chemokines and T cell infiltration in the development of kidney disease. As chemokine receptor blockade inches toward clinical use, the field remains an attractive area with potential for unexpected opportunity in the future.
Subject(s)
Chemokines/physiology , Kidney Diseases/etiology , Acute Kidney Injury/etiology , Acute Kidney Injury/immunology , Animals , Chemokines/genetics , Chronic Disease , Gene Expression Regulation , Humans , Immunity, Innate , Receptors, Chemokine/physiology , T-Lymphocytes/immunology , T-Lymphocytes, Regulatory/physiology , Th17 Cells/physiologyABSTRACT
TGF-ß/Smad signaling plays a role in fibrogenesis, but therapies targeting TGF-ß are ineffective in treating renal fibrosis. Here, we explored the therapeutic potential of targeting TGF-ß-induced microRNA in the progression of renal fibrosis. Microarray analysis and real-time PCR revealed upregulation of miR-21 in tubular epithelial cells (TECs) in response to TGF-ß. Lack of Smad3, but not lack of Smad2, prevented cells from upregulating miR-21 in response to TGF-ß. In addition, Smad3-deficient mice were protected from upregulation of miR-21 and fibrosis in the unilateral ureteral obstruction model. In contrast, conditional knockout of Smad2 enhanced miR-21 expression and renal fibrosis. Furthermore, ultrasound-microbubble-mediated gene transfer of a miR-21-knockdown plasmid halted the progression of renal fibrosis in established obstructive nephropathy. In conclusion, these data demonstrate that Smad3, but not Smad2, signaling increases expression of miR-21, which promotes renal fibrosis. Inhibition of miR-21 may be a therapeutic approach to suppress renal fibrosis.