Xiao-Ru Huang

The Protective Role of Smad7 in Diabetic Kidney Disease: Mechanism and Therapeutic Potential

OBJECTIVE Although Smad3 has been considered as a downstream mediator of transforming growth factor-β (TGF-β) signaling in diabetes complications, the role of Smad7 in diabetes remains largely unclear. The current study tests the hypothesis that Smad7 may play a protective role and has therapeutic potential for diabetic kidney disease. RESEARCH DESIGN AND METHODS Protective role of Smad7 in diabetic kidney disease was examined in streptozotocin-induced diabetic mice that have Smad7 gene knockout (KO) and in diabetic rats given Smad7 gene transfer using an ultrasound-microbubble-mediated technique. RESULTS We found that mice deficient for Smad7 developed more severe diabetic kidney injury than wild-type mice as evidenced by a significant increase in microalbuminuria, renal fibrosis (collagen I, IV, and fibronectin), and renal inflammation (interleukin-1β [IL-1β], tumor necrosis factor-α [TNF-α], monocyte chemoattractant protein-1 [MCP-1], intracellular adhesion molecule-1 [ICAM-1], and macrophages). Further studies revealed that enhanced renal fibrosis and inflammation in Smad7 KO mice with diabetes were associated with increased activation of both TGF-β/Smad2/3 and nuclear factor-κB (NF-κB) signaling pathways. To develop a therapeutic potential for diabetic kidney disease, Smad7 gene was transferred into the kidney in diabetic rats by an ultrasound-microbubble-mediated technique. Although overexpression of renal Smad7 had no effect on levels of blood glucose, it significantly attenuated the development of microalbuminuria, TGF-β/Smad3-mediated renal fibrosis such as collagen I and IV and fibronectin accumulation and NF-κB/p65-driven renal inflammation including IL-1β, TNF-α, MCP-1, and ICAM-1 expression and macrophage infiltration in diabetic rats. CONCLUSIONS Smad7 plays a protective role in diabetic renal injury. Overexpression of Smad7 may represent a novel therapy for the diabetic kidney complication.

Interleukin-1 induces tubular epithelial-myofibroblast transdifferentiation through a transforming growth factor-β1-dependent mechanism in vitro

Interleukin-1 (IL-1) has been shown to exert profibrotic activity in a number of disease models, including crescentic glomerulonephritis and pulmonary fibrosis, but the mechanisms by which this operates are poorly understood. Recent studies have identified a novel mechanism promoting renal fibrosis: tubular epithelial-myofibroblast transdifferentiation (TEMT). The present study examined whether IL-1 can stimulate TEMT in vitro. Cells of the normal rat kidney tubular epithelial cell line (NRK52E) were grown to confluence on collagen-coated plates and cultured for 5 days in the presence 1 to 20 ng/mL of IL-1alpha. Doses of 10 to 20 ng/mL of IL-1 caused transdifferentiation of NRK52E cells into myofibroblast-like cells. Scanning electron microscopy identified IL-1-induced morphological changes as a loss of apical-basal polarity and microvilli, cell hypertrophy, and the development of an elongated and invasive appearance. Phenotypically, IL-1-induced TEMT was characterized by de novo messenger RNA and protein expression of the mesenchymal marker alpha-smooth muscle actin, shown by Northern blotting, immunohistochemistry, and Western blotting. This was accompanied by loss of the epithelial marker E-cadherin. The addition of an excess of IL-1-receptor antagonist completely inhibited IL-1-induced TEMT. IL-1 was shown to stimulate the secretion of active transforming growth factor-beta1 (TGF-beta1) by NRK52E cells. Furthermore, the addition of a neutralizing anti-TGF-beta1 antibody inhibited IL-1-induced TEMT. In conclusion, IL-1 is a profibrogenic cytokine capable of inducing TEMT through a TGF-beta1-dependent mechanism. This may represent a novel mechanism by which IL-1 induces renal fibrosis in vivo.

miR-29b as a Therapeutic Agent for Angiotensin II-induced Cardiac Fibrosis by Targeting TGF-β/Smad3 signaling

Loss of miR-29 is associated with cardiac fibrosis. This study examined the role and therapeutic potential of miR-29 in mouse model of hypertension induced by angiotensin II (AngII). By using microRNA microarray, in situ hybridization, and real-time polymerase chain reaction, we found that AngII-induced cardiac fibrosis in the hypertensive heart and in cultured cardiac fibroblasts were associated with downregulation of miR-29a-c via a Smad3-dependent mechanism. In vitro knockdown of miR-29b enhanced but overexpression of miR-29b inhibited AngII-induced fibrosis, revealing a protective role of miR-29b in cardiac fibrosis in response to AngII. This was further demonstrated in vivo by the ability of overexpressing miR-29b in the mouse heart to prevent AngII-mediated cardiac fibrosis and cardiac dysfunction. Importantly, we also found that restored miR-29b in the established hypertensive heart was capable of blocking progressive cardiac fibrosis and improving cardiac dysfunction, demonstrating a therapeutic potential of miR-29b for chronic heart disease. Further studies revealed that targeting the transforming growth factor (TGF)-β1 coding sequence region, thereby inhibiting TGF-β/Smad3 signaling, could be a new mechanism by which miR-29b inhibited AngII-induced cardiac fibrosis. In conclusion, miR-29b plays a protective role in AngII-mediated cardiac remodeling and may be a therapeutic agent for cardiac fibrosis by targeting the TGF-β/Smad3 pathway.

Disruption of Smad4 impairs TGF-β/Smad3 and Smad7 transcriptional regulation during renal inflammation and fibrosis in vivo and in vitro

The mechanism by which TGF-β regulates renal inflammation and fibrosis is largely unclear; however, it is well accepted that its biological effects are mediated through Smad2 and Smad3 phosphorylation. Following activation, these Smads form heteromeric complex with Smad4 and translocate into the nucleus to bind and regulate the expression of target genes. Here we studied the roles of Smad4 to regulate TGF-β signaling in a mouse model of unilateral ureteral obstruction using conditional Smad4 knockout mice and in isolated Smad4 mutant macrophages and fibroblasts. Disruption of Smad4 significantly enhanced renal inflammation as evidenced by a greater CD45(+) leukocyte and F4/80(+) macrophage infiltration and upregulation of IL-1β, TNF-α, MCP-1, and ICAM-1 in the obstructed kidney and in IL-1β-stimulated macrophages. In contrast, deletion of Smad4 inhibited renal fibrosis and TGF-β1-induced collagen I expression by fibroblasts. Further studies showed that the loss of Smad4 repressed Smad7 transcription, leading to a loss of functional protein. This, in turn, inhibited IκBα expression but enhanced NF-κB activation, thereby promoting renal inflammation. Interestingly, deletion of Smad4 influenced Smad3-mediated promoter activities and the binding of Smad3 to the COL1A2 promoter, but not Smad3 phosphorylation and nuclear translocation, thereby inhibiting the fibrotic response. Thus, Smad4 may be a key regulator for the diverse roles of TGF-β1 in inflammation and fibrogenesis by interacting with Smad7 and Smad3 to influence their transcriptional activities in renal inflammation and fibrosis.

Loss of angiotensin-converting enzyme 2 enhances TGF-β/Smad-mediated renal fibrosis and NF-κB-driven renal inflammation in a mouse model of obstructive nephropathy.

It is known that angiotensin (Ang)-converting enzyme (ACE) 2 catalyzes Ang II to Ang 1–7 to prevent the detrimental effect of Ang II on blood pressure, renal fibrosis, and inflammation. However, mechanisms of renoprotective role of Ace2 remain largely unclear. The present study tested the hypothesis that deficiency of Ace2 may accelerate intrarenal Ang II-mediated fibrosis and inflammation independent of blood pressure in a model of unilateral ureteral obstructive (UUO) nephropathy induced in Ace2+/y and Ace2−/y mice. Results showed that both Ace2+/y and Ace2−/y mice had normal levels of blood pressure and plasma Ang II/Ang 1–7. In contrast, deletion of ACE2 resulted in a fourfold increase in the ratio of intrarenal Ang II/Ang 1–7 in the UUO nephropathy. These changes were associated with the development of more intensive tubulointerstitial fibrosis (α-SMA, collagen I) and inflammation (TNF-α, IL-1β, MCP-1, F4/80+ cells, and CD3+T cells) in Ace2−/y mice at day 3 (all P<0.05) after UUO, becoming more profound at day 7 (all P<0.01). Enhanced renal fibrosis and inflammation in the UUO kidney of Ace2−/y mice were largely attributed to a marked increase in the intrarenal Ang II signaling (AT1-ERK1/2 mitogen-activated protein kinase), TGF-β/Smad2/3, and NF-κB signaling pathways. Further studies revealed that enhanced TGF-β/Smad and NF-κB signaling in the UUO kidney of Ace2−/y mice was associated with upregulation of an E3 ligase Smurf2 and a loss of renal Smad7. In conclusion, enhanced Ang II-mediated TGF-β/Smad and NF-κB signaling may be the mechanisms by which loss of Ace2 enhances renal fibrosis and inflammation. Smad7 ubiquitin degradation mediated by Smurf2 may be a central mechanism by which Ace2−/y mice promote TGF-β/Smad2/3-mediated renal fibrosis and NF-κB-driven renal inflammation in a mouse model of UUO nephropathy.

Diverse roles of TGF-β receptor II in renal fibrosis and inflammation in vivo and in vitro.

TGF‐β1 binds receptor II (TβRII) to exert its biological activities but its functional importance in kidney diseases remains largely unclear. In the present study, we hypothesized that TβRII may function to initiate the downstream TGF‐β signalling and determine the diverse role of TGF‐β1 in kidney injury. The hypothesis was examined in a model of unilateral ureteral obstructive (UUO) nephropathy and in kidney fibroblasts and tubular epithelial cells in which the TβRII was deleted conditionally. We found that disruption of TβRII inhibited severe tubulointerstitial fibrosis in the UUO kidney, which was associated with the impairment of TGF‐β/Smad3 signalling, but not with the ERK/p38 MAP kinase pathway. In contrast, deletion of TβRII enhanced NF‐κB signalling and renal inflammation including up‐regulation of Il‐1β and Tnfα in the UUO kidney. Similarly, in vitro disruption of TβRII from kidney fibroblasts or tubular epithelial cells inhibited TGF‐β1‐induced Smad signalling and fibrosis but impaired the anti‐inflammatory effect of TGF‐β1 on IL‐1β‐stimulated NF‐κB activation and pro‐inflammatory cytokine expression. In conclusion, TβRII plays an important but diverse role in regulating renal fibrosis and inflammation. Impaired TGF‐β/Smad3, but not the non‐canonical TGF‐β signalling pathway, may be a key mechanism by which disruption of TβRII protects against renal fibrosis. In addition, deletion of TβRII also enhances NF‐κB signalling along with up‐regulation of renal pro‐inflammatory cytokines, which may be associated with the impairment of anti‐inflammatory properties of TGF‐β1. Copyright

Kidney-targeting Smad7 gene transfer inhibits renal TGF-β/MAD homologue (SMAD) and nuclear factor κB (NF-κB) signalling pathways, and improves diabetic nephropathy in mice

Aims/hypothesisThe TGF-β/MAD homologue (SMAD) and nuclear factor κB (NF-κB) signalling pathways have been shown to play a critical role in the development of renal fibrosis and inflammation in diabetic nephropathy. We therefore examined whether targeting these pathways by a kidney-targeting Smad7 gene transfer has therapeutic effects on renal lesions in the db/db mouse model of type 2 diabetes.MethodsWe delivered Smad7 plasmids into the kidney of db/db mice using kidney-targeting, ultrasound-mediated, microbubble-inducible gene transfer. The histopathology, ultrastructural pathology and pathways of TGF-β/SMAD2/3-mediated fibrosis and NF-κB-dependent inflammation were evaluated.ResultsIn this mouse model of type 2 diabetes, Smad7 gene therapy significantly inhibited diabetic kidney injury, compared with mice treated with empty vectors. Symptoms inhibited included: (1) proteinuria and renal function impairment; (2) renal fibrosis such as glomerular sclerosis, tubulo-interstitial collagen matrix abundance and renal inflammation, including Inos (also known as Nos2), Il1b and Mcp1 (also known as Ccl2) upregulation, as well as macrophage infiltration; and (3) podocyte and endothelial cell injury as demonstrated by immunohistochemistry and/or electron microscopy. Further study demonstrated that the improvement of type 2 diabetic kidney injury by overexpression of Smad7 was associated with significantly inhibited local activation of the TGF-β/SMAD and NF-κB signalling pathways in the kidney.Conclusions/interpretationOur results clearly demonstrate that kidney-targeting Smad7 gene transfer may be an effective therapy for type 2 diabetic nephropathy, acting via simultaneous modulation of the TGF-β/SMAD and NF-κB signalling pathways.

Disruption of Smad7 Promotes ANG II-Mediated Renal Inflammation and Fibrosis via Sp1-TGF-β/Smad3-NF.κB-Dependent Mechanisms in Mice

Smad7 is an inhibitory Smad and plays a protective role in obstructive and diabetic kidney disease. However, the role and mechanisms of Smad7 in hypertensive nephropathy remains unexplored. Thus, the aim of this study was to investigate the role and regulatory mechanisms of Smad7 in ANG II-induced hypertensive nephropathy. Smad7 gene knockout (KO) and wild-type (WT) mice received a subcutaneous infusion of ANG II or control saline for 4 weeks via osmotic mini-pumps. ANG II infusion produced equivalent hypertension in Smad7 KO and WT mice; however, Smad7 KO mice exhibited more severe renal functional injury as shown by increased proteinuria and reduced renal function (both p<0.05) when compared with Smad7 WT mice. Enhanced renal injury in Smad7 KO mice was associated with more progressive renal fibrosis with elevated TGF-β/Smad3 signalling. Smad7 KO mice also showed more profound renal inflammation including increased macrophage infiltration, enhanced IL-1β and TNF-α expression, and a marked activation of NF-κB signaling (all p<0.01). Further studies revealed that enhanced ANG II-mediated renal inflammation and fibrosis in Smad7 KO mice were also associated with up-regulation of Sp1 but downregulation of miR-29b expression. Taken together, the present study revealed that enhanced Sp1-TGF-β1/Smad3-NF-κB signaling and loss of miR-29 may be mechanisms by which deletion of Smad7 promotes ANG II-mediated renal fibrosis and inflammation. Thus, Smad7 may play a protective role in ANG II-induced hypertensive kidney disease.

Expression of Macrophage Migration Inhibitory Factor in Acute Ischemic Myocardial Injury

Macrophage migration inhibitory factor (MIF) is a key mediator in inflammatory or immune-mediated diseases, although its role in heart diseases is unknown. This study investigated the expression of MIF in the myocardium in the development of acute myocardial infarction (AMI). By use of immunohistochemistry, Western blotting, RT-PCR, and in situ hybridization, the gene and protein expression of MIF in the heart at 6 hr, 1 day, 3 days, 1 week, and 2 weeks after AMI was studied. In both normal and sham-operated rats, MIF mRNA and protein were expressed constitutively at low levels by the myocytes. By contrast, MIF mRNA was rapidly upregulated by the surviving myocytes in the infarcted region and, to a lesser extent, the non-infarcted region, accounting for a sevenfold increase at 6 hr after AMI (p<0.001). This was followed by a fourfold increase in MIF protein expression at day 1 after AMI (p<0.05). Macrophages were found accumulated in the infarcted region, being significant at day 1 (p<0.01) and progressive increased over the 2-week time course (p<0.01) in which MIF was found expressed in these cells. The results indicated that the infiltrating macrophages and myocytes were sources of MIF in the infarcted region. The latter cells became activated and involved in the amplification of inflammatory response in AMI. Therefore, upregulation of myocardial MIF may contribute to macrophage accumulation in the infarcted region and their pro-inflammatory role may participate in the myocyte damage seen in AMI.

MicroRNA-29b inhibits peritoneal fibrosis in a mouse model of peritoneal dialysis

TGF-β/Smad3 signaling plays a pivotal role in the pathogenesis of peritoneal fibrosis associated with peritoneal dialysis (PD). MicroRNA-29 (miR-29) is known as a potent downstream inhibitor of TGF-β/Smad3 in renal fibrosis. In this study, we examined the therapeutic potential for miR-29b on PD-related peritoneal fibrosis in a mouse model of PD induced by daily infusion of 4.25% dextrose-containing PD fluid (PDF). MiR-29b-expressing plasmid was delivered into the peritoneum via an ultrasound-microbubble-mediated system before and at day 14 after PDF. We found that mice on PD developed peritoneal fibrosis with impaired peritoneal function, which was associated with a loss of miR-29b. In contrast, overexpression of miR-29b before the PDF infusion showed a protective effect on peritoneal fibrosis including EMT and prevented peritoneal dysfunction. Moreover, delayed miR-29b treatment until peritoneal fibrosis was established at day 14 also halted the progression of peritoneal fibrosis at day 28. Further studies identified that blockade of the Sp1-TGF-β/Smad3 pathway may be a mechanism by which miR-29b inhibited peritoneal fibrosis. In conclusion, treatment with miR-29b may represent a novel and effective therapy for PD-associated peritoneal fibrosis.