miR-192 Mediates TGF-β/Smad3-Driven Renal Fibrosis

TGF-β/Smad3 promotes renal fibrosis, but the mechanisms that regulate profibrotic genes remain unclear. We hypothesized that miR-192, a microRNA expressed in the kidney may mediate renal fibrosis in a Smad3-dependent manner. Microarray and real-time PCR demonstrated a tight association between upregulation of miR-192 in the fibrotic kidney and activation of TGF-β/Smad signaling. Deletion of Smad7 promoted miR-192 expression and enhanced Smad signaling and fibrosis in obstructive kidney disease. In contrast, overexpression of Smad7 to block TGF-β/Smad signaling inhibited miR-192 expression and renal fibrosis in the rat 5/6 nephrectomy model; in vitro, overexpression of Smad7 in tubular epithelial cells abolished TGF-β1–induced miR-192 expression. Furthermore, Smad3 but not Smad2 mediated TGF-β1–induced miR-192 expression by binding to the miR-192 promoter. Last, overexpression of a miR-192 mimic promoted and addition of a miR-192 inhibitor blocked TGF-β1–induced collagen matrix expression. Taken together, miR-192 may be a critical downstream mediator of TGF-β/Smad3 signaling in the development of renal fibrosis.TGF-β is a profibrogenic cytokine that mediates renal fibrosis positively by activating its downstream mediators called Smad2 and Smad3 but negatively by its inhibitory factor Smad7^1–4; however, the mechanisms as to how the Smads regulate the fibrogenic genes during renal fibrosis remain unclear.MicroRNAs (miRNAs) are small, noncoding RNAs with approximately 22 nucleotides. The mature miRNAs can complementarily bind to the mRNA 3′ untranslated region to regulate the gene expression by translational repression or induction of mRNA degradation.⁵ Increasing evidence shows that TGF-β may act by regulating miRNAs to exhibit its biological effects such as epithelial-to-mesenchymal transition (EMT),⁵ suggesting that TGF-β regulates the expression of these miRNAs to promote EMT.Several miRNAs, including miR-192, -194, -204, -215, and -216, are highly expressed in the kidney, as compared with other organs.^6,7 In the context of renal fibrosis, expression levels of miR-192 increased significantly in glomeruli isolated from diabetic mice.⁸ In vitro, miR-192 is induced by TGF-β1 and mediates TGF-β–induced collagen expression in mesangial cells (MCs) by downregulating ZEB2 expression.⁸ In contrast, a recent study also found that TGF-β1 suppresses miR-192 expression in human tubular epithelial cells (TEC) and loss of miR-192 promotes fibrogenesis in diabetic nephropathy.⁹ The discrepancy in these two studies with opposite findings and understanding of miR-192 in diabetic nephropathy necessitates further investigation of the potential role of miR-192 and the mechanisms that regulate miR-192 expression during renal fibrosis under various disease conditions. Thus, this study tested the hypothesis that TGF-β1 may act by stimulating Smad3 to regulate miR-192 expression during renal fibrosis. This was tested in rodent models of obstructive and remnant kidney diseases induced in mice that lacked Smad3 or Smad7, had conditional knockout (KO) for Smad2, or overexpressed renal Smad7. In addition, TGF-β–induced miR-192 expression via the Smad3-dependent mechanism was determined in TECs overexpressing Smad7 or knocking down for Smad2 or Smad3 and in Smad2 or Smad3 KO mouse embryonic fibroblasts (MEFs).     

TGF-β1 Promotes Lymphangiogenesis during Peritoneal Fibrosis

Peritoneal fibrosis (PF) causes ultrafiltration failure (UFF) and is a complicating factor in long-term peritoneal dialysis. Lymphatic reabsorption also may contribute to UFF, but little is known about lymphangiogenesis in patients with UFF and peritonitis. We studied the role of the lymphangiogenesis mediator vascular endothelial growth factor-C (VEGF-C) in human dialysate effluents, peritoneal tissues, and peritoneal mesothelial cells (HPMCs). Dialysate VEGF-C concentration correlated positively with the dialysate-to-plasma ratio of creatinine (D/P Cr) and the dialysate TGF-β1 concentration. Peritoneal tissue from patients with UFF expressed higher levels of VEGF-C, lymphatic endothelial hyaluronan receptor-1 (LYVE-1), and podoplanin mRNA and contained more lymphatic vessels than tissue from patients without UFF. Furthermore, mesothelial cell and macrophage expression of VEGF-C increased in the peritoneal membranes of patients with UFF and peritonitis. In cultured mesothelial cells, TGF-β1 upregulated the expression of VEGF-C mRNA and protein, and this upregulation was suppressed by a TGF-β type I receptor (TGFβR-I) inhibitor. TGF-β1–induced upregulation of VEGF-C mRNA expression in cultured HPMCs correlated with the D/P Cr of the patient from whom the HPMCs were derived (P<0.001). Moreover, treatment with a TGFβR-I inhibitor suppressed the enhanced lymphangiogenesis and VEGF-C expression associated with fibrosis in a rat model of PF. These results suggest that lymphangiogenesis associates with fibrosis through the TGF-β–VEGF-C pathway.The decrease in ultrafiltration capacity that is associated with the high peritoneal solute transport that is observed after prolonged peritoneal dialysis (PD) treatment is a major reason for its discontinuation.^1–4 Several studies have shown that a higher peritoneal solute transport rate is associated with reduced survival of PD patients.^1,2,5 The characteristic features of chronic peritoneal damage in PD treatment are associated with submesothelial fibrosis and neoangiogenesis.^6,7 Analyses of the surface peritoneum showed no significant changes in vessel density with duration of PD.^6,8 In addition, the vessel density in patients with ultrafiltration failure (UFF) was significantly higher than the vessel density in normal individuals or non-PD patients, but it was not higher than the vessel density in patients undergoing PD.⁶ These findings suggest that factors other than increased vascular density may be involved in disease states associated with increased transport of peritoneal membranes. In addition, the relationship between peritoneal fibrosis and UFF remains obscure.Blood capillaries have a continuous basal lamina with tight interendothelial junctions and are supported by pericytes and smooth muscle cells. In contrast, lymphatic capillaries are thin-walled with a wide lumen and do not contain pericytes or basement membrane. The structures of lymphatic vessels are suitable for the removal of tissue fluid, cells, and macromolecules from the interstitium.^9–11 If lymphangiogenesis develops in the peritoneal membrane, absorption of the PD fluid could be increased and lead to UFF. An increase in the number of lymphatic vessels has recently been reported in several disease conditions, including tumor metastasis,^12–15 chronic respiratory inflammatory diseases,^16–18 wound healing,¹⁹ and renal transplant rejection.^20,21 We recently reported that lymphangiogenesis had developed in tubulointerstitial fibrosis of human renal biopsy specimens,²² and we also reported the mechanisms of lymphangiogenesis in rat unilateral ureteral obstruction models.²³The lymphatic absorption rate, which is measured by the rate at which intraperitoneally administered radioactive serum albumin or macromolecule dextran 70 disappears, is significantly higher in patients with UFF, and lymphatic reabsorption is considered to be one of the causes of UFF.^24–27 However, the results from these clinical approaches have been controversial.^28,29 In addition, little is known about the pathology and the process of lymphangiogenesis in patients with UFF and peritonitis.In this study, we investigated lymphangiogenesis and the expression of vascular endothelial growth factor-C (VEGF-C), which is a potentially important mediator of lymphangiogenesis, in human peritoneal tissues, PD effluent, and peritoneal mesothelial cells. We also explored VEGF-C induction by TGF-β1 in the human mesothelial cell line (Met-5A) and cultured human peritoneal mesothelial cells (HPMCs) from the spent PD effluent of patients with varying rates of peritoneal transport. Finally, we explored the relationship between peritoneal fibrosis and lymphangiogenesis in rats that were administered chlorhexidine gluconate (CG) into the abdominal cavity, which provides a model of chemically induced peritoneal inflammation/fibrosis.^30–32 This work is the first report to show that lymphangiogenesis is linked to the peritoneal fibrosis that is often associated with a high peritoneal transport rate.     

Estrogen inhibits vaginal tropoelastin and TGF-β1 production

Introduction and hypothesis

The aim of this study was to quantify the effects of estrogen on vaginal smooth muscle cell (SMC) tropoelastin and transforming growth factor (TGF)-β1 production.

Methods

Primary SMC were incubated with estradiol, and cell proliferation was assessed by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay at 48 h. Supernatants were collected and tropoelastin and TGF-β1 levels measured.

Results

SMC proliferation was significantly increased by estradiol [relative cell number, mean ± standard error (SE), estradiol 0.1 μM 116?±?19 % of control (P?=?NS), 1 μM 127?±?13 % of control (P?<?0.05), 10 μM 153?±?26 % of control, (P?<?0.05)]. Tropoelastin production was significantly decreased by estrogen [mean ± SE, estradiol 0.1 μM 78?±?2 % of control (P?<?0.05), 1 μM 76?±?4 % of control (P?<?0.05), 10 μM 67?±?3 % of control, (P?<?0.05)]. In addition, TGF-β1 production was significantly decreased [mean ± SE, estradiol 0.1 μM 96?±?4 % of control (P?=?NS), 1 μM 84?±?6 % of control (P?<?0.05), 10 μM 70?±?6 % of control, (P?<?0.05)].

Conclusion

Estrogen increases vaginal SMC proliferation and inhibits tropoelastin and TGF-β1 production.     

NAD(P)H Oxidase Mediates TGF-β1–Induced Activation of Kidney Myofibroblasts

TGF-β1 expression closely associates with activation and conversion of fibroblasts to a myofibroblast phenotype and synthesis of an alternatively spliced cellular fibronectin variant, Fn-ED-A. Reactive oxygen species (ROS), such as superoxide, which is a product of NAD(P)H oxidase, also promote the transition of fibroblasts to myofibroblasts, but whether these two pathways are interrelated is unknown. Here, we examined a role for NAD(P)H oxidase–derived ROS in TGF-β1–induced activation of rat kidney fibroblasts and expression of α-smooth muscle actin (α-SMA) and Fn-ED-A. In vitro, TGF-β1 stimulated formation of abundant stress fibers and increased expression of both α-SMA and Fn-ED-A. In addition, TGF-β1 increased both the activity of NADPH oxidase and expression of Nox2 and Nox4, homologs of the NAD(P)H oxidase family, indicating that this growth factor induces production of ROS. Small interfering RNA targeted against Nox4 markedly inhibited TGF-β1–induced stimulation of NADPH oxidase activity and reduced α-SMA and Fn-ED-A expression. Inhibition of TGF-β1 receptor 1 blocked Smad3 phosphorylation; reduced TGF-β1–enhanced NADPH oxidase activity; and decreased expression of Nox4, α-SMA, and Fn-ED-A. Diphenyleneiodonium, an inhibitor of flavin-containing enzymes such as the Nox oxidases, had no effect on TGF-β1–induced Smad3 but reduced both α-SMA and Fn-ED-A protein expression. The Smad3 inhibitor SIS3 reduced NADPH oxidase activity, Nox4 expression, and blocked α-SMA and Fn-ED-A, indicating that stimulation of myofibroblast activation by ROS is downstream of Smad3. In addition, TGF-β1 stimulated phosphorylation of extracellular signal–regulated kinase (ERK1/2), and this was inhibited by blocking TGF-β1 receptor 1, Smad3, or the Nox oxidases; ERK1/2 activation increased α-SMA and Fn-ED-A. Taken together, these results suggest that TGF-β1–induced conversion of fibroblasts to a myofibroblast phenotype involves a signaling cascade through Smad3, NAD(P)H oxidase, and ERK1/2.Progression of renal fibrosis involves expansion of interstitial myofibroblasts and extracellular matrix accumulation, resulting in the loss of function and ultimately renal failure.^1,2 The origin of myofibroblasts is under extensive investigation, and evidence indicates the cells may be derived from several sources, including an expansion of activated resident fibroblasts, perivascular adventitial cells, blood-borne stem cells that migrate into the glomerular mesangial or interstitial compartment, or tubular epithelial-to-mesenchymal transition and migration into the peritubular interstitial space. Regardless of their origin, there is common agreement that the myofibroblast is the cell most responsible for interstitial expansion and matrix accumulation during the course of renal fibrosis. TGF-β1 is the predominant growth factor responsible for matrix synthesis by mesenchymal cells such as fibroblasts in vitro and during renal fibrosis.^3,4 Indeed, there is a close correlation in the cellular expression of TGF-β1, a fibroblast transition to an activated, α-smooth muscle actin (α-SMA)-positive myofibroblast phenotype, and synthesis of an alternatively spliced isoform of fibronectin, Fn-ED-A.⁵ TGF-β1 differentially regulates the expression of Fn-ED-A in fibroblasts^6–8 and induces expression of α-SMA in a variety of mesenchymal cells in culture.^9,10 Indeed, a functional ED-A domain is mandatory for α-SMA induction by TGF-β1.^7,8,10 Moreover, TGF-β1 is frequently associated with a myofibroblast phenotype in liver, lung, and kidney disease,^1,11–13 and all three proteins frequently co-localize in these disease settings. In addition, a co-localization of α-SMA and Fn-ED-A is frequently observed in fibrotic disease as well as in glomerular and interstitial lesions in kidney diseases previously investigated in our laboratory.^14–17Accumulating evidence also indicates that reactive oxygen species (ROS), mainly in the form of superoxide, play a significant role in the initiation and progression of cardiovascular^18,19 and renal^20–25 disease. ROS are involved in distinct cell functions, including hypertrophy, migration, proliferation, apoptosis, and regulation of extracellular matrix.^25–28 More specific, the NAD(P)H oxidases of the Nox family have gained heightened attention as mediators of injury associated with vascular diseases, including hypertension, atherosclerosis, heart disease, and diabetes.^18,19,29,30 NAD(P)H oxidase generation of superoxide is recognized as an important mediator of cell proliferation in glomerulonephritis²² and matrix accumulation in diabetic nephropathy^25,31–33 and fibrosis.^21,24 Adventitial fibroblasts are also a major source of superoxide in the aorta,^19,34–36 therefore being highly relevant to renal disease. This is because the renal perivascular space is noticeably reactive and is the site where myofibroblasts may first appear during the course of renal disease and fibrosis.^17,37–39The observations that both TGF-β1 and ROS induce fibroblasts to α-SMA–positive myofibroblast phenotype^40–42 suggest that these two pathways are interrelated and may share signaling pathways in kidney disease. TGF-β signaling occurs through a well-established process involving two downstream pathways: Smad and extracellular signal–regulated kinase (ERK).^43–45 TGF-β/Smad signaling (Smad2 and Smad3) is tightly controlled by mitogen-activated protein kinase (MAPK; ras/MEK/ERK) signaling cascades.⁴⁶ A regulatory role for ROS in PDGF and angiotensin II–induced signal transduction has gained recognition^47,48; however, a role for ROS in TGF-β signaling is less well understood. It is also unknown whether kidney myofibroblasts express NAD(P)H oxidase homologs or generate ROS in response to TGF-β1. Given TGF-β1–induced myofibroblast activation and matrix synthesis during renal disease may be linked to ROS, we examined a role for NAD(P)H oxidase in TGF-β1–induced Smad3 and ERK signaling as well as kidney myofibroblast activation, as assessed by a switch to an α-SMA–positive phenotype and expression of Fn-ED-A expression in vitro.     

Smad2 Protects against TGF-β/Smad3-Mediated Renal Fibrosis

Smad2 and Smad3 interact and mediate TGF-β signaling. Although Smad3 promotes fibrosis, the role of Smad2 in fibrogenesis is largely unknown. In this study, conditional deletion of Smad2 from the kidney tubular epithelial cells markedly enhanced fibrosis in response to unilateral ureteral obstruction. In vitro, Smad2 knockdown in tubular epithelial cells increased expression of collagen I, collagen III, and TIMP-1 and decreased expression of the matrix-degrading enzyme MMP-2 in response to TGF-β1 compared with similarly treated wild-type cells. We obtained similar results in Smad2-knockout fibroblasts. Mechanistically, Smad2 deletion promoted fibrosis through enhanced TGF-β/Smad3 signaling, evidenced by greater Smad3 phosphorylation, nuclear translocation, promoter activity, and binding of Smad3 to a collagen promoter (COL1A2). Moreover, deletion of Smad2 increased autoinduction of TGF-β1. Conversely, overexpression of Smad2 attenuated TGF-β1–induced Smad3 phosphorylation and collagen I matrix expression in tubular epithelial cells. In conclusion, in contrast to Smad3, Smad2 protects against TGF-β–mediated fibrosis by counteracting TGF-β/Smad3 signaling.TGF-β/Smad signaling has been shown to play a critical role in renal fibrosis.^1–4 It is now clear that TGF-β1 signals through the heteromeric complex of TGF-β type I receptor and TGF-β type II receptor to activate two key downstream mediators, Smad2 and Smad3, to exert its biological activities such as cell growth, differentiation, extracellular matrix (ECM) production, and apoptosis.⁵ Although it is known that Smad2 and Smad3 physically interact and are structurally similar with >90% homology in their amino acid sequences,⁶ the distinct functions of these two genes in embryonic development has been noted. Genetic deletion of Smad2 in mice results in embryonic lethality at an early stage of development, whereas mice null for Smad3 survive with impaired immunity.^7,8 Although the functional role of Smad3 in cell growth, differentiation, apoptosis, tissue repair and fibrosis, and immune responses has been well studied,^8–10 the pathophysiologic role of Smad2 in these processes remains largely unclear. This may be attributed to the unavailability of Smad2 knockout (KO) mice for such studies as a result of the early embryonic lethality.In the context of tissue repair and fibrosis, Smad3 has been studied extensively, but little attention has been paid to the functional role of Smad2. It is now well accepted that Smad3 is a key mediator of TGF-β signaling in ECM production and tissue fibrosis. This may be associated with the finding that Smad3-binding elements are found in most collagen promoters^11–13; therefore, TGF-β1–induced collagen matrix expression is Smad3 dependent. Emerging evidence has shown that Smad3 plays an important role in tissue repair and fibrosis including wound healing,¹⁴ epithelial-to-mesenchymal transition (EMT),¹⁵ and tissue scar formation under various disease conditions in skin,¹³ lung,¹⁶ heart,¹⁷ kidney,¹⁵ and liver¹⁸; however, the functional importance of Smad2 and the interaction between Smad2 and 3 in the profibrotic response to TGF-β1 remain largely unclear. Thus, this study investigated the functional role of Smad2 in ECM production and renal fibrosis in vivo in a mouse model of unilateral ureteral obstruction (UUO) and in vitro in tubular epithelial cells (TECs) with knockdown or overexpression of Smad2 and in mouse embryonic fibroblasts (MEFs) lacking Smad2. The mechanisms of Smad2 in regulating fibrosis in response to TGF-β1 were explored.     

BAMBI Elimination Enhances Alternative TGF-β Signaling and Glomerular Dysfunction in Diabetic Mice

BMP, activin, membrane-bound inhibitor (BAMBI) acts as a pseudo-receptor for the transforming growth factor (TGF)-β type I receptor family and a negative modulator of TGF-β kinase signaling, and BAMBI^−/− mice show mild endothelial dysfunction. Because diabetic glomerular disease is associated with TGF-β overexpression and microvascular alterations, we examined the effect of diabetes on glomerular BAMBI mRNA levels. In isolated glomeruli from biopsies of patients with diabetic nephropathy and in glomeruli from mice with type 2 diabetes, BAMBI was downregulated. We then examined the effects of BAMBI deletion on streptozotocin-induced diabetic glomerulopathy in mice. BAMBI^−/− mice developed more albuminuria, with a widening of foot processes, than BAMBI^+/+ mice, along with increased activation of alternative TGF-β pathways such as extracellular signal–related kinase (ERK)1/2 and Smad1/5 in glomeruli and cortices of BAMBI^−/− mice. Vegfr2 and Angpt1, genes controlling glomerular endothelial stability, were downmodulated in glomeruli from BAMBI^−/− mice with diabetes. Incubation of glomeruli from nondiabetic BAMBI^+/+ or BAMBI^−/− mice with TGF-β resulted in the downregulation of Vegfr2 and Angpt1, effects that were more pronounced in BAMBI^−/− mice and were prevented by a MEK inhibitor. The downregulation of Vegfr2 in diabetes was localized to glomerular endothelial cells using a histone yellow reporter under the Vegfr2 promoter. Thus, BAMBI modulates the effects of diabetes on glomerular permselectivity in association with altered ERK1/2 and Smad1/5 signaling. Future therapeutic interventions with inhibitors of alternative TGF-β signaling may therefore be of interest in diabetic nephropathy.     

TGF-β1 gene polymorphisms and primary vesicoureteral reflux in childhood

The aim of this study was to assess the association between the transforming growth factor-β1 (TGF-β1) gene polymorphisms rs1800469 (commonly known as T-509C) and rs1982073 (commonly known as Leu ¹⁰→Pro) and primary vesicoureteral reflux (VUR) and renal scarring. Using a case–control approach, we examined 121 children with primary VUR and 169 controls. Genotyping of the TGF-β1 gene polymorphisms was performed by restriction fragment length polymorphism (RFLP) analysis. The ^99mTc-DMSA– or ^99mTc-unitiol–single photon emission computed tomography method was used to evaluate renal scars in 84 of 121 VUR children. Statistical analysis revealed differences in rs1800469 genotype frequencies between VUR patients and controls (p = 0.0021). Our data demonstrate that individuals homozygous for the TT genotype are at risk of primary VUR [odds ratio (95% confidence interval) = 2.7 (1.46–5.08)]. Distribution of the rs1982073 polymorphism was similar in VUR children and controls. In terms of renal scarring, patients were stratified into non-scar and scar subgroups, and no differences in the genotype frequencies of either polymorphism was found. Previous reports have shown that the TT genotype of the rs1800469 polymorphism is a risk factor for renal scarring in primary VUR, and the results of our study suggest that this same polymorphism is associated with susceptibility to this congenital uropathy.     

TGF-β Signaling via TAK1 Pathway: Role in Kidney Fibrosis

In progressive kidney diseases, fibrosis represents the common pathway to end-stage kidney failure. Transforming growth factor-β1 (TGF-β1) is a pleiotropic cytokine that has been established as a central mediator of kidney fibrosis. Emerging evidence shows a complex scheme of signaling networks that enable multifunctionality of TGF-β1 actions. Specific targeting of the TGF-β signaling pathway is seemingly critical and an attractive molecular therapeutic strategy. TGF-β1 signals through the interaction of type I and type II receptors to activate distinct intracellular pathways involving the Smad and the non-Smad. The Smad signaling axis is known as the canonical pathway induced by TGF-β1. Importantly, recent investigations have shown that TGF-β1 also induces various non-Smad signaling pathways. In this review, we focus on current insights into the mechanism and function of the Smad-independent signaling pathway via TGF-β-activated kinase 1 and its role in mediating the profibrotic effects of TGF-β1.     

Lipoxins Attenuate Renal Fibrosis by Inducing let-7c and Suppressing TGFβR1

Lipoxins, which are endogenously produced lipid mediators, promote the resolution of inflammation, and may inhibit fibrosis, suggesting a possible role in modulating renal disease. Here, lipoxin A4 (LXA₄) attenuated TGF-β1–induced expression of fibronectin, N-cadherin, thrombospondin, and the notch ligand jagged-1 in cultured human proximal tubular epithelial (HK-2) cells through a mechanism involving upregulation of the microRNA let-7c. Conversely, TGF-β1 suppressed expression of let-7c. In cells pretreated with LXA₄, upregulation of let-7c persisted despite subsequent stimulation with TGF-β1. In the unilateral ureteral obstruction model of renal fibrosis, let-7c upregulation was induced by administering an LXA₄ analog. Bioinformatic analysis suggested that targets of let-7c include several members of the TGF-β1 signaling pathway, including the TGF-β receptor type 1. Consistent with this, LXA₄-induced upregulation of let-7c inhibited both the expression of TGF-β receptor type 1 and the response to TGF-β1. Overexpression of let-7c mimicked the antifibrotic effects of LXA₄ in renal epithelia; conversely, anti-miR directed against let-7c attenuated the effects of LXA₄. Finally, we observed that several let-7c target genes were upregulated in fibrotic human renal biopsies compared with controls. In conclusion, these results suggest that LXA₄-mediated upregulation of let-7c suppresses TGF-β1–induced fibrosis and that expression of let-7c targets is dysregulated in human renal fibrosis.There is a growing appreciation of the role of endogenously produced lipid mediators including lipoxins, resolvins, and PGD synthase metabolites in promoting the resolution of inflammatory responses.^1–4 We and others recently described distinct proresolution and antifibrotic properties of lipoxins in renal fibrosis.^5,6 TGF-β1 is implicated in numerous fibrotic conditions including tubulointerstitial fibrosis and diabetic kidney disease. The development of fibrosis in this context may reflect activation of parenchymal fibroblasts, recruitment of circulating fibrocytes, and de-differentiation of epithelia and pericytes.^7,8 In this study, we investigated the effect of lipoxin A4 (LXA₄) on TGF-β1–induced fibrotic responses of renal epithelial cells and the mechanism underlying attenuation of this fibrotic injury pattern by lipoxins.MicroRNAs (miRNAs) comprise a class of small noncoding RNAs that negatively regulate gene expression by base-pairing to partially complementary sites in the 3′ untranslated regions (UTRs) of target mRNA, preventing translation. miRNAs are implicated in the development and progression of a wide range of complex human diseases,^9–12 including diabetic nephropathy.^13–15 Here we report that LXA₄ upregulates expression of the miRNA let-7c in HK-2 cells and attenuates response to TGF-β1. Lipoxins are protective in unilateral ureteral obstruction (UUO)–induced renal fibrosis and we report that this is associated with increased let-7c expression. Conversely, TGF-β1 decreases let-7c expression and this is associated with increased expression of let-7c targets, including TGFβ receptor type 1 (TGFβR1), collagens (COL1A1, COL1A2), and thrombospondin (THBS1). Overexpression of let-7c mimics the fibrosuppressant effects of LXA₄, whereas suppression of let-7c mimics responses to TGF-β1. The importance of regulation of let-7c targets is further supported by evidence from human renal biopsy material where we report upregulation of several let-7c targets in CKD.     

大鼠肝星状细胞TGF-β3/TGF-β1mRNA比值与TGF-β1、MMP-9、TIMP-1表达的关系

目的 探讨大鼠肝星状细胞(HSC-T6)中转化生长因子-β3(TGF-β3)和转化生长因子-β1(TGF-β1)mRNA比值变化与TGF-β1、MMP-9、TIMP-1表达的关系.方法 构建质粒pcDNA 3.1(+)-TGF-β3和pcDNA 3.1(+)-TGF-β1.将pcDNA 3.1(+)-TGF-1β1转染HSC-T6细胞株,经筛选建立高表达TGF-β1的HSC-T6细胞阳性克隆.pcDNA 3.1(+)-TGF-β3转染该阳性克隆,48 h后荧光定量PCR法和Western blot法分别检测TGF-β3、TGF-β1、MMP-9和TIMP-1 mRNA和蛋白的变化.结果 空白组、对照组、阳性克隆组及TGF-β3干预组中,TGF-β3/TGF-β1mRNA比值分别为0.286±0.070、0.874±0.141、0.448±0.327和1.277±0.244;阳性克隆组与空白组和对照组相比,TGF-β1和TIMP-1的mRNA及蛋白表达明显增高(P<0.05),MMP-9的mRNA及蛋白表达明显减少(P<0.05);TGF-β3干预组与阳性克隆组相比,TGF-β1蛋白和TIMP-1 mRNA及蛋白表达明显下降(P<0.05),MMP-9 mRNA及蛋白表达明显增加(P<0.05).结论 TGF-β3能下调TGF-β1蛋白表达;当TGF-β3/TGF-β1mRNA比值>1时,TGF-β1和TIMP-1表达减少,MMP-9表达增加;当TGF-β3/TGF-β1mRNA比值<1时,TGF-β1表达减少,TIMP-1和MMP-9表达无变化.     

Autophagy Regulates TGF-β Expression and Suppresses Kidney Fibrosis Induced by Unilateral Ureteral Obstruction

Autophagy is an evolutionarily conserved process that cells use to degrade and recycle cellular proteins and remove damaged organelles. During the past decade, there has been a growing interest in defining the basic cellular mechanism of autophagy and its roles in health and disease. However, the functional role of autophagy in kidney fibrosis remains poorly understood. Here, using GFP-LC3 transgenic mice, we show that autophagy is induced in renal tubular epithelial cells (RTECs) of obstructed kidneys after unilateral ureteral obstruction (UUO). Deletion of LC3B (LC3^−/− mice) resulted in increased collagen deposition and increased mature profibrotic factor TGF-β levels in obstructed kidneys. Beclin 1 heterozygous (beclin 1^+/−) mice also displayed increased collagen deposition in the obstructed kidneys after UUO. We also show that TGF-β1 induces autophagy in primary mouse RTECs and human renal proximal tubular epithelial (HK-2) cells. LC3 deficiency resulted in increased levels of mature TGF-β in primary RTECs. Under conditions of TGF-β1 stimulation and autoinduction, inhibition of autolysosomal protein degradation by bafilomycin A1 increased mature TGF-β protein levels without alterations in TGF-β1 mRNA. These data suggest a novel intracellular mechanism by which mature TGF-β1 protein levels may be regulated in RTECs through autophagic degradation, which suppresses kidney fibrosis induced by UUO. The dual functions of TGF-β1, as an inducer of TGF-β1 autoinduction and an inducer of autophagy and TGF-β degradation, underscore the multifunctionality of TGF-β1.In the kidney, fibrosis is responsible for chronic progressive kidney failure, and the prevalence of CKD is increasing worldwide.^1,2 Extracellular matrix (ECM) protein production and progressive accumulation are hallmarks of renal tubulointerstitial fibrosis in progressive kidney disease. Collagens are the main components of the ECM in the kidney, and type I collagen (Col-I) is the major type associated with disease states.^3,4 The cellular mechanisms that facilitate tubulointerstitial fibrosis after injury remain incompletely defined. Recent lineage tracing or genetic fate mapping studies have strongly challenged the theory that renal tubular epithelial cells (RTECs) traverse the tubular basement membrane to become myofibroblasts in a process of epithelial-to-mesenchymal transition (EMT), but rather, that interstitial pericytes/perivascular fibroblasts are the myofibroblast progenitor cells.^5–7 It also has been proposed that profibrotic factors, such as TGF-β1, are upregulated in the tubular interstitial area on injury, leading to kidney fibrosis.⁸ TGF-β1 induces production of ECM proteins, including fibronectin and collagens, and inhibits degradation of ECM proteins mainly by matrix metalloproteinases.^9–11 Given the recent evidence that casts doubts about the role of EMT in vivo, how RTECs contribute to the development of renal tubulointerstitial fibrosis is not entirely clear.TGF-β is synthesized as a single polypeptide precursor that includes a preregion signal peptide, which is removed by proteolytic cleavage, and pro–TGF-β, containing a proregion called the latency-associated peptide and a mature TGF-β, and it converts to homodimeric pro–TGF-β through disulfide bonds.¹² After cleavage by proprotein convertases, such as furin, latency-associated peptide remains noncovalently associated with the dimeric form of mature TGF-β as the small latent complex (SLC).¹³ SLC formation occurs in the Golgi apparatus, and mature TGF-β is secreted as part of SLC and associated with latent TGF-β–binding protein to form TGF-β large latent complex, which interacts with ECM. On stimulus, the dimeric form of mature TGF-β is dissociated from large latent complex and becomes the bioactive mature TGF-β ligand, which can then bind TGF-β receptors to trigger downstream Smad-dependent or -independent signaling pathways.^12,13 Thus, the availability of mature TGF-β is the limiting factor of TGF-β activity and not TGF-β synthesis per se, because the body generates more pro–TGF-β than necessary. Whereas TGF-β/TGF-β receptor downstream signaling pathways have been extensively investigated, the regulation of TGF-β maturation and bioavailability has not been well studied but may serve as an important target for fibrotic diseases that alter TGF-β signaling.Macroautophagy, hereafter referred to as autophagy, is a fundamental cellular homeostatic process that cells use to degrade and recycle cellular proteins and remove damaged organelles. The process of autophagy involves the formation of double membrane–bound vesicles called autophagosomes that envelop and sequester cytoplasmic components, including macromolecular aggregates and cellular organelles, for bulk degradation by a lysosomal degradative pathway.¹⁴ Autophagy can be induced in response to either intracellular or extracellular factors, such as amino acid or growth factor deprivation, hypoxia, low cellular energy state, endoplasmic reticulum stress or oxidative stress, organelle damage, and pathogen infection.^15–22 To date, over 30 genes involved in autophagy have been identified in yeast, and they have been termed autophagy-related genes (Atgs). The mammalian ortholog of Atg8 is comprised of a family of proteins known as microtubule-associated protein 1 light chain 3 (LC3) that functions as a structural component in the formation of autophagosomes.²³ LC3B (herein referred to as LC3) is the best characterized form and the most widely used as an autophagic marker. The conversion of the cytosolic form of LC3 (LC3-I) to lipidated form (LC3-II) indicates autophagosome formation. In contrast to LC3, Beclin 1, encoded by the beclin 1 gene, is the mammalian ortholog of yeast Atg6 that is required for the initiation of autophagy through its interaction with Vps34. Homozygous deletion of beclin 1 (beclin 1^−/−) exhibits early embryonic lethality, whereas heterozygous deletion (beclin 1^+/−) results in increased incidence of spontaneous tumorigenesis, abnormal proliferation of mammary epithelial cells and germinal center B lymphocytes, and increased susceptibility to neurodegeneration.^24–27We previously reported that autophagy promotes intracellular degradation of Col-I induced by TGF-β1 in glomerular mesangial cells.²⁸ In the present study, we explored the functional role of autophagy in an in vivo model of progressive kidney fibrosis induced by unilateral ureteral obstruction (UUO) in autophagy-deficient LC3 null (LC3^−/−) and heterozygous (beclin 1^+/−) mice and green fluorescent protein (GFP)-LC3 transgenic mice. We also performed functional studies in primary cultured mouse RTECs and human renal proximal tubular epithelial (HK-2) cells. We hypothesized that induction of autophagy in RTECs promotes TGF-β degradation and thereby reduces TGF-β secretion and suppresses development of kidney fibrosis.     

TGF-β/BMP Pathways and the Podocyte

Renal fibrosis is the major determinant in progression of acute and chronic kidney diseases. Transforming growth factor-β (TGF-β) has been shown to be an important mediator of progressive fibrosis. Several studies have implicated that TGF-β1 is involved in the tight balance of survival and apoptotic responses in podocytes that are Smad-dependent or independent. Bone morphogenic protein-7 (BMP-7), another member of the TGF-β superfamily, has to date been involved primarily in kidney development and was described as an active blocker of TGF-β-induced profibrotic effects. Here, we summarize the direct effects of these two cytokines on podocytes. We describe their involvement in podocyte survival and apoptosis pathways with the potential to modify the critical steps in podocyte apoptosis induction. Our group has analyzed the cross-talk of BMP-7 and TGF-β1 signaling in podocytes and we describe BMP-7 as a cytoprotective factor that could antagonize proapoptotic TGF-β signals. In addition, we identified various extracellular and intracellular modifiers that can influence this sensitive cross-talk. On the basis of our work and the work of others we conclude that the balance of TGF-β1 and BMP-7 signaling and involvement of extracellular and intracellular modifiers in these cascades are important parts of podocyte physiology and pathophysiology.     

胰腺癌组织smad4mRNA,TGF-β1和TGF-βR1的表达及意义

目的研究胰腺癌组织中smad4mRNA,TGF-β1和TGF-βR1的表达及其生物学意义.方法采用原位杂交方法检测癌及癌旁组织的smad4mRNA,免疫组化方法检测TGF-β1和TGF-βR1.结果 53例胰腺癌smad4mRNA,TGF-β1和TGF-βR1表达阳性率明显地低于25例癌旁上皮(均P＜0.05).高分化腺癌smad4mRNA,TGF-β1和TGF-βR1表达阳性率明显高于低分化腺癌(P＜0.05～0.01),无转移癌明显高于转移癌(P＜0.05～0.01).三者在胰腺癌中表达阳性率均存在密切相关系.结论 smad4mRNA,TGF-β1和TGF-βR1表达与胰腺癌的发生发展、生物学行为和预后有密切关系,检测三者表达水平对早期发现胰腺癌及指导临床治疗和评估预后可能有较重要价值.     

Tests of linkage and/or association of TGF-β1 and COL1A1 genes with bone mass

Transforming growth factor beta 1 (TGF-1) is involved in bone metabolism and collagen type I alpha 1 (COL1A1) is the most abundant protein of bone matrix. Both have been considered as candidate genes for osteoporosis. In this study, we employed the transmission disequilibrium test (TDT) to examine the relationship between each of the two genes with bone mineral density (BMD) and bone mineral content (BMC) at the spine and hip in a sample of 1668 subjects from 387 Caucasian nuclear families. For the TGF-1 gene, three SNPs, SNP1, SNP2, and SNP4 (located in exon 1, intron 4 and intron 5, respectively) were tested and the minor allele frequencies were 30.9%, 2.1% and 27.0%, respectively. All eight possible haplotypes (TGF1–8) were observed. For the COL1A1 gene, the minor allele frequencies of SNP5, SNP6 and SNP8 (located in exon 1, intron 1, and exon 45, respectively) were 15.2%, 18.7%, 2.0%, respectively, and only six of eight potential haplotypes (COL1–6) were obtained. In the whole sample, total associations were observed between haplotype COL5 with spine BMD (P=0.027), haplotypes COL3 and TGF4 with hip BMC (P=0.002, 0.003, respectively). Within-family associations were found for spine BMD at haplotypes TGF4 (P=0.027) in female offspring families and TGF3 (P=0.021) in male offspring families. Further studies with denser markers and larger sample size are required to eventually define the relationship between these two genes with bone mass at the spine and hip.